Tolcndv resistant melon plants

ABSTRACT

The present invention relates to ToLCNDV resistant melon plants comprising one or two introgression fragments in their genome.

The application concerns cultivated melon plants (Cucumis melo)resistant to infection with tomato leaf curl New Dehli virus (ToLCNDV).The resistant melon plants have a genomic introgression fragment onchromosome 11 and/or on chromosome 12. The introgression fragment onchromosome 11 comprises a QTL which confers resistance to ToLCNDV in arecessive manner. The introgression fragment on chromosome 12 comprisesa QTL which confers resistance to ToLCNDV in a partially dominantmanner. Both QTLs together result in cultivated melon plants which showvery few (average disease score of at least 8) or even no symptoms(average disease score of 9). Also disclosed are markers for identifyingplants or plant parts comprising those introgression fragments andmethods for identifying or producing ToLCNDV resistant melon plants.

Tomato leaf curl New Dehli virus (ToLCNDV) is classified as Begomovirusbelonging to the family Geminiviridae. ToLCNDV has a bipartite genomeconsisting of two single stranded DNA molecules referred to as DNA A andDNA B (Saez et al., 2016, Annals of Applied Biology).

ToLCNDV was initially found to infect tomato (Solanum lycopersicum)plants in 1995 in India. Later ToLCNDV was found to infect also plantsof other Solanacea species, like Solanum melongena (aubergine), chilipepper (Capsicum spp.) and Solanum tuberosum (potato). In 2012 infectionof curcubit species (courgette, Cucurbita pepo var. giromontiina) byToLCNDV was found in Spain and in 2015 the virus was identified as thedisease source in melon, cucumber and courgette in Tunisia. In themeantime, infection of many Curcubitacea species such as Benincasahispida (wax gourd), Citrullus lanatus (watermelon), Cucumis melo(melon), Cucumis melo var. flexuosus (snake melon), Cucumis sativus(cucumber), Cucurbita moschata (musky gourd), Cucurbita pepo (pumpkin),Cucurbita pepo var. giromontiina (courgette), Lagenaria siceraria(bottle gourd), Luffa cylindrica (sponge gourd), Momordica charantia(bitter gourd) have been proven. Infection of weeds (e.g. Ecliptaprostrata—Asteraceae) and other crops such as Hibiscus cannabinus(kenaf—Malvaceae) and Carica papaya (papaya—Caricaceae) was alsoreported. In the Mediterranean region the disease does occur in variouscrop species in Italy (Sicilia), Spain and Tunisia. In Asian countries,infection was proven in different crops in Bangladesh, India, Indonesia,Pakistan, Philippines, Sri Lanka, Taiwan and Thailand. Furtherinformation on geographical distribution of ToLCNDV is lacking, but fromthe observations made today, the virus clearly seems to further spreadgeographically as well as to other crops.

Disease symptoms in general comprise phenotypic appearance of yellowmosaic on leaves, leaf curling, vein swelling, and plant stunting.Cucurbits upon infection of young plants with ToLCNDV show stuntedgrowth and decreased or suppressed fruit production. Also fruits showingskin roughness and longitudinal cracking have been reported. Thus,ToLCNDV causes economic losses in various important crop species and isa major threat. Infection of plants by ToLCNDV occurs persistently bytransmission of the virus by the phloem sucking whitefly (Bemisiatabaci). (European and Mediterranean Plant Protection Organization, EPPORS 2015/114, 2016/024, 2016/040, Entry date 2015-06).

In sponge gourd resistance to ToLCNDV has been shown to be controlled bya single dominant gene (Islam et al., 2010, Euphytica 174(1):83-89).

In tomato, transgenic plants resistant to ToLCNDV have been produced bysilencing virus genes (Varma & Praveen, 2006, ISB News Report).

Assays for transmission of ToLCNDV by mechanically transferring the sapof an infected zucchini plant to non-infected plants from other cucurbitgenera (Cucumis, Cucurbita, Citrullus, Lagenaria) have been developed.Five Cucumis melo subsp. agrestis accessions (subsp. agrestis var.momordica: Mom-Khalnd/Kharbuja, Mom-PI124Ind/PI124112,Mom-PI124Ind/PI414723 and subsp. agrestis wild types: Ag-WM9Ind/WM9,Ag-WM7Ind/WM7) resistant to ToLCNDV were identified (Lopez et al., 2015,Euphytica 204(3), 679-691). Confirmation of resistance of theseaccessions by using the natural whitefly infection system was notperformed.

Saez et al. 2016, pages 214-216 (Proceedings of Cucurbitaeceae 2016, theXIth EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae Jul.24-28 2016, Warsaw, Poland) describe that ToLCNDV resistance of the C.melo subsp. agrestis accession WM-7 segregated in an F2 population in a3:1 ratio of tolerant:susceptible, suggesting a single dominantresistance gene being present in WM-7. As in Lopez et al. 2015, supra,mechanical inoculation was used, and not natural infection via whiteflyvector. In a later publication (Saez et al., 14 Jul. 2017, Plant CellReport, DOI 10.1007/s00299-017-2175-3), the resistance from WM-7 wasmapped and found to be conferred by a dominant QTL on chromosome 11.

WO2017/114848 describe a ToLCNDV tolerance conferring QTL of a C. melosubsp. agrestis var. acidulous donor (referred to as H-MLCND-32) mappedto chromosome 11, which is recessive. A mix of two lines containing theQTL (RIL-69 and RIL-82) were deposited under accession number NCIMB42506. According to Table 5, the QTL must lie in the sub-region betweenSEQ ID NO: 7 (Melon_sbg_33761_74) and SEQ ID NO: 16(Melon_sbg_16835_17), because RIL-30 has the resistance phenotype, butlacks the donor SNP markers for SEQ ID NO: 1 (Melon_sbg_617_42) to 6(Melon_sbg_60684_74). Three SNPs were found to have the most predictivevalue for the phenotype, namely Melon_sbg_33761_74 (SEQ ID NO: 7),Melon_sbg_2720_78 (SEQ ID NO: 8) and Melon_sbg_14207_58 (SEQ ID NO: 9)(see page 13, line 29-30 and FIG. 2). The donor is described to vary insymptom levels, e.g. of 7 plants, 4 had no symptoms, 2 had moderateyellowing symptoms and 1 was completely symptomatic (Table 1).

Various Cucurbita species (C. pepo, C. moschata, C. maxima, C.fraternal, C. ficifolia) have been screened for resistance to ToLCNDV bythe mechanical sap transmission screening assay. Four Curcubita moschataaccessions (PI 604506, PI 381814, Nigerian local, Kurokawa) were foundto show low symptoms upon, mechanical infection with ToLCNDV. Thisresult however could be reproduced upon whitefly infection only for twoof the accession (PI 604506, PI 381814), demonstrating that resistanceto ToLCNDV should be tested not only under artificial conditions but inaddition by using the naturally occurring whitefly infection system(Saez et al., 2016, Annals of Applied Biology). Therefore, it is unclearif the accessions identified to be resistant to infection by ToLCNDV areresistant under natural growing conditions when the virus is transferredby whiteflies.

Attempts to control ToLCNDV infection of crop plants comprise vector(whitefly) control by insecticides applications and adaption of culturalpractices, including use of virus free crop material (transplants),establishment of crop free periods, weed management (eliminating virusinfected weeds) and destruction of infected plants in the field.However, because of the complex epidemiological factors associated withthe disease, these attempts are not always effective (Saez et al., 2016,Annals of Applied Biology).

Therefore, there is a need to establish further measurements forreducing ToLCNDV infections, further spreading of the virus to othergeographical areas and spreading to other crop species. Breeding ofvarieties resistant to ToLCNDV would be essential for managing thedisease.

An object of the present invention is to provide measurements for thecontrol of ToLCNDV infection in cultivated melon plants.

The present invention discloses cultivated melon plant cells and melonplants being resistant or even highly resistant to infections byToLCNDV.

It is commonly known that ToLCNDV does infect various different plantspecies of the Curcubitaceae species, including melon species. It isalso well known that ToLCNDV is transmitted persistently from infectedplants to non-infected plants by the plant sucking pest Bemisia tabaci(whitefly). Transfer of ToLCNDV from one crop species to different cropspecies or even from weed species to crop species has been demonstrated.Whiteflies may pick up ToLCNDV from outside the controlled area evenfrom different species and transfer it to melon plants grown in thecontrolled area. Whitefly vector control, therefore, is of limitedeffectiveness for preventing ToLCNDV infection. ToLCNDV resistant melonplants have the advantage that they would withstand infection withToLCNDV without major yield losses, even if plants around the area wherethe melon plants are grown are infected with ToLCNDV.

The inventors found a ToLCNDV resistant donor accession of C. melosubsp. melo, obtained from France but, according to the descriptor,originating from western India, and have mapped the resistance in asegregating population, using whitefly infection with a Spanish ToLCNDVstrain for resistance phenotyping. Two Quantitative Trait Loci (QTLs)were identified, one on chromosome 11 (QTL11) and one on chromosome 12(QTL12) and introgressed from the donor accession into an elite Piel deSapo melon line and into an elite Galia melon line. Seeds of the Piel deSapo line, comprising both QTL11 and QTL12 introgressions in homozygousform, were deposited by Nunhems B. V. under Accession number NCIMB42625on Aug. 10, 2016 in accordance with the Budapest Treaty. In these seedsthe donor genotype for the Single Nucleotide Polymorphism (SNP) markers(SNP_01 to SNP_07) provided herein is present. The donor itself is notuniform and has very long (elongated) fruits with a narrow fruitdiameter and a low brix. It also has other negative characteristics,such as a very weak attachment of the peduncle at maturity, a very shortshelf life of the fruits, etc., i.e. it is not an accession of agronomicvalue. By identifying and transferring QTL11 and/or QTL12 from the donorinto cultivated melon, it is now possible to make cultivated melonvarieties and cultivars of high agronomique value (with uniformcharacteristics and marketable fruits having high brix and good shelflife) with resistance against ToLCNDV and thus it is possible tocultivate those melon varieties in ToLCNDV infested areas without yieldloss.

“Melon plant cells” or “melon plants” or “cultivated melon plants orcells” also designated as muskmelon plant cells or muskmelon plants inthe art shall be understood in context with the present invention to beplant cells originating from the species Cucumis melo or to be plantsbelonging to the species Cucumis melo. Cucumis melo, can be classifiedinto: C. melo var. cantalupensis, C. melo var. inodorous and C. melovar. reticulatus. C. melo var. cantalupensis are also referred to asCantaloupes and are primarily round in shape with prominent ribs andalmost no netting. Most have orange, sweet flesh and they are usuallyvery fragrant. In contrast to the European cantaloupe, the NorthAmerican ‘Cantaloupe’ is not of this type, but belongs to the truemuskmelons. C. melo var. inodorous (or winter melons) can be subdividedinto different types, such as Honeydew melon, Piel de Sapo, Sugar melon,Japanese melon, etc. C. melo var. reticulatus is the true muskmelon,with reticulated skin (netted) and includes Galia melons, Sharlyn melonsand the North American cantaloupe.

Cultivated melon and the wild relatives of melon is/are diploid andhas/have 12 pairs of homologous chromosomes, numbered 1 to 12.

“Resistant” or “being resistant to” shall be understood in context ofthe present invention to mean a plant which is a host species of aparticular pathogen and can therefore be infected by a given pathogen,but wherein the plant comprises a genetic element (e.g. an introgressionfragment) resulting in reduction of pathogen growth and/or spreading inthe plant after infection compared to the susceptible plant lacking thegenetic element. In context of the present invention “resistant” or“being resistant to” in particular refers to plant cells or plants beingresistant to ToLCNDV. Resistance is a relative term which can span arange of (different) reactions in the plant cell or plant, triggered bypathogen infection. The effect of those reactions by the plant cell orplant can be measured by various means. Typically the effect is measuredby defining a symptom level appearing in the plant cell or plant.Typically average symptoms (average disease score) of several plants ofa line (e.g. 10 or more) are compared to average symptoms (averagedisease score) of several plants of a control line or variety,preferably a susceptible control line or variety. Thus at least 10 ormore individual plants of a line or variety are scored at one time pointand the average disease score is calculated. Concerning the presentinvention, the following commonly known symptom levels (or diseasescore) are applied according to phenotypic observations taken afterToLCNDV infection:

1=Dead plant

2=Severe mosaic and curling, chlorosis and growth reduction. No recovery

3=Strong mosaic and curling, chlorosis and growth reduction. No recovery

4=Curling and mosaic, chlorosis, no or mild growth reduction. Norecovery

5=Curling and mosaic, chlorosis, no growth reduction. Slight recovery ofthe upper plant zone

6=Mild curling, mosaic and chlorosis, no growth reduction. Recovery ofthe upper middle plant

7=Mild curling, mosaic and chlorosis, no growth reduction. Symptomsappear only in the lower plant zone

8=Faint mosaic

9=No symptoms

For determining the symptom level (or disease score) preferably youngplants are infected with ToLCNDV. Young plants are preferably plantshaving the age of the first true leaf being expanded, preferablyapproximately 12-15 days after sowing. Infection is preferably carriedout via feeding of the vector (Bemisia) carrying the virus. For thispurpose plants are germinated and grown under optimal or close tooptimal conditions. The symptom level is preferably determined at leastonce at e.g. 30 days after infection (or later, e.g. 31, 32, 33, 34, 35days after infection). Optionally symptom level is determined twice oreven three times at different time-points following infection to confirmthe result, e.g. a first scoring at approximately 15, 20 or 25 daysafter infection and a second scoring at approximately 30 days afterinfection (or later, e.g. 31, 32, 33, 34, 35 days after infection) withToLCNDV. See also the Examples. The average disease score is calculatedfor each line or variety at each time point. In one aspect a plant lineor variety is said to be “resistant” against ToLCNDV infection if it hasan average disease score of 7.0 or higher, while the susceptible controlline or variety, such as variety Gandalf or Vedantrais, has an averagedisease score of 4.0 or less, 3.0 or less or 2.0 or less, when grownunder the same conditions and infected in the same way. In anotheraspect a plant line or variety is said to be “highly resistant” againstToLCNDV infection if it has an average disease score of 8.0 or higher,preferably if it has an average disease score of 9.0, while thesusceptible control line or variety, such as variety Gandalf (or GandalfF1, Nunhems) or Vedantrais, has an average disease score of 4.0 or less,3.0 or less or 2.0 or less, when grown under the same conditions andinfected in the same way.

It has been observed that introgressions of specific fragments locatedon chromosome 11 (comprising QTL11) and 12 (comprising QTL12) from thesame wild melon plant donor into cultivated melon plants confersresistance to ToLCNDV infection in cultivated melon plants or cellsderived therefrom. While the fragment on chromosome 11 needs to bepresent in homozygous form to confer resistance, it is sufficient thatthe fragment on chromosome 12 is present only in the heterozygous statefor conferring ToLCNDV resistance, demonstrating that the fragment onchromosome 11 confers resistance in a recessive manner, while thefragment on chromosome 12 confers resistance to ToLCNDV infection in apartially dominant manner. Single Nucleotide Polymorphisms (SNPs) onchromosome 11 and 12 were identified which are closely linked to thefragment of chromosome 11 and 12 conferring ToLCNDV resistance. The SNPgenotype of the resistant donor (i.e. the nucleotide of theintrogression fragment) is present in homozygous form in the depositedseeds, i.e. the donor genotype is present in homozygous form for SNP_01to SNP_04 (linked to QTL11) and also for SNP_05 to SNP_07 (linked toQTL12). The SNPs can, therefore, be used to test the presence of theintrogression fragment comprising the QTL11 or QTL12 in a plant cell,plant tissue, plant part, and/or in marker assisted selection (MAS) totransfer the QTLs into other elite melon lines or varieties. The SNPscan also be used to select plants comprising smaller introgressionsfragments than the fragments present in the deposited seeds, whereby thesmaller sub-fragments retain the QTL. Alternatively the SNPs can be usedto identify other donors which comprise QTL11 and/or QTL12 and tointrogress these QTLs into cultivated melon.

The present invention, therefore, relates in one aspect to cultivatedmelon plant cells or melon plants (or plant parts) comprising anintrogression fragment from chromosome 11 and/or 12 of a ToLCNDVresistant donor plant, wherein the introgression fragment comprises thesequence of the donor plant in-between SNP_01 and SNP 04 for thefragment on chromosome 11, preferably in-between SNP_02 and SNP_04 forthe fragment on chromosome 11 or optionally in-between SNP_03 andSNP_04; or optionally in-between SNP_01 and SNP_03, or in-between SNP_02and SNP_03, or in-between SNP_01 and SNP_02; and/or in-between SNP_05and SNP_07 for the fragment on chromosome 12, optionally in-betweenSNP_05 and SNP_06, or in-between SNP_06 and SNP_07. The ToLCNDVresistance conferring QTL is present on the introgression fragment, ascan be determined by a resistance assay as described herein.

In one aspect the present invention relates to cultivated melon plantcells or melon plants (or plant parts) comprising an introgressionfragment from chromosome 11 and/or 12 of a ToLCNDV resistant donorplant, wherein the introgression fragment confers ToLCNDV resistance andthe introgression fragment is detectable by (comprises) the SNP genotypeof the donor plant for one or more (or all) of the following SNPs:SNP_01, SNP_02, SNP_03 and/or SNP_04, and optionally any SNP in-betweenSNP_01 and SNP_04, for the fragment on chromosome 11; and/or wherein theintrogression fragment confers ToLCNDV resistance and the introgressionfragment is detectable by (comprises) the SNP genotype of the donorplant for one or more (or all) of the following SNPs: SNP_05, SNP_06and/or SNP_07, and optionally any SNP in-between SNP_05 and SNP_07, forthe fragment on chromosome 12. The ToLCNDV resistance conferring QTL ispresent on the introgression fragment as can be determined by aresistance assay as described herein.

In one aspect the SNP genotype for SNP_01, SNP_02, SNP_03 and/or SNP_04is the SNP genotype of the introgression donor of QTL11, i.e. an Adenine(A) at nucleotide 101 of SEQ ID NO: 1 (SNP_01), a Guanine (G) atnucleotide 101 of SEQ ID NO: 2 (SNP_02), a Thymine (T) at nucleotide 101of SEQ ID NO: 3 (SNP_03), a Thymine (T) at nucleotide 101 of SEQ ID NO:4 (SNP_04).

In one aspect the plant, plant part or plant cell comprises QTL11 andcomprises the SNP donor genotype for at least SNP_03, as this SNP ismost significantly associated with the ToLCNDV resistance QTL11. In afurther aspect the plant, plant part or plant cell comprises the SNPdonor genotype for at least SNP_03 and SNP_04, or for at least SNP_03and SNP_02. Optionally, the plant, plant part or plant cell comprisesQTL11 and comprises the SNP donor genotype for SNP_01, SNP_02 andSNP_03; or for SNP_02, SNP_03 and SNP_04. In one aspect the plant, plantpart or plant cell comprises QTL11 comprises the SNP donor genotype forall four SNPs (SNP_01, SNP_02, SNP_03 and SNP_04).

In one aspect the SNP genotype for SNP_05, SNP_06 and/or SNP_07, is theSNP genotype of the introgression donor of QTL12, i.e. a C (Cytosine) atnucleotide 101 of SEQ ID NO: 5 (SNP_05) and/or a Guanine (G) atnucleotide 101 of SEQ ID NO: 6 (SNP_06), and optionally a Thymine (T) atnucleotide 101 of SEQ ID NO: 7 (SNP_07). In one aspect the plant, plantpart or plant cell comprises QTL12 and comprises the SNP donor genotypefor at least SNP_06, as this SNP is most significantly associated withthe ToLCNDV resistance QTL12. In a further aspect the plant, plant partor plant cell comprises the SNP donor genotype for at least SNP_05 andSNP_06, or for at least SNP_06 and SNP_07. Optionally, the plant, plantpart or plant cell comprises QTL12 and comprises the SNP donor genotypefor SNP_05 and SNP_06 and optionally also for SNP_07.

When referring herein to the introgression fragment comprising the donorchromosome sequence or a donor sequence (and the QTL) “in-between” twoSNPs (Single Nucleotide Polymorphisms), this encompasses in one aspectthat one or both of the two SNPs themselves are also from the resistantdonor, i.e. have the donor nucleotide. Thus, regarding the QTL onchromosome 11, SNP_01, SNP_02, SNP_03 and SNP_04 may all have theresistant donor genotype. Or only SNP_01 and SNP_02 may have theresistant donor genotype; or only SNP_02 and SNP_03 may have theresistant donor genotype; or only SNP_03 and SNP_04 may have theresistant donor genotype. Or only a single SNP, i.e. only SNP_01, oronly SNP_02 or only SNP_03, or only SNP_04 has the resistant donorgenotype. The SNPs that do not have the resistant donor genotype thushave another genotype, the recipient genotype. The recipient genotypefor a SNP may be any of the other 3 nucleotides, i.e. for SNP_01 therecipient genotype may be Cytosine, Guanine or Thymine.

Thus, for example when stating that the introgression fragment isin-between SNP_05 and SNP_06 regarding the QTL on chromosome 12, SNP_05and SNP_06 may both have the resistant donor genotype. Or only a singleSNP, i.e. only SNP_05 or only SNP_06 may have the resistant donorgenotype. Optionally also SNP_07 may have the resistant donor genotype.

The reason that not all of the SNPs provided herein need to have theresistant donor genotype is that the introgression fragment comprisingthe QTL from the donor may be smaller than the chromosome fragmentintrogressed e.g. in the deposited seeds, but the fragment stillcomprises the QTL11 or QTL12. Still, a plant can be recognized tocontain the introgression fragment (comprising the QTL11 or 12) by thephenotype, and/or by transferring the fragment to a susceptible plantand thereby transferring the ToLCNDV resistance phenotype, or bysequencing the region between the SNP markers to identify the donorfragment, or other methods known to the skilled person, such assaturating the region with more SNP markers, allelism tests, identifyingthe causal gene, etc.

Thus, a combination of methods can be used to show that the QTL11 orQTL12 is present in a plant cell or plant, even if not for all of thelinked SNPs the donor SNP genotype is present. QTL11 confers an averageToLCNDV resistance of at least 7.0 when transferred into a susceptibleline or variety and is recessive. QTL12 confers an average ToLCNDVresistance score of at least 5.0 or 6.0 when transferred into asusceptible line or variety and is partially dominant. And when bothQTLs are combined in a cultivated plant so that their phenotype isexpressed, an average ToLCNDV resistance score of at least 8.0 or atleast 9.0 is obtained.

When QTL12 is transferred into a melon plant which already has somelevel of ToLCNDV resistance, it increases the average resistance levelby 1 or 2 scores, so e.g. if the recipient plant has an averageresistance level of 7.0, introduction of QTL12 will increase theresistance level to at least 8.0 or 9.0.

Thus, in one aspect the SNP genotype of the melon plant comprising theintrogression fragment on chromosome 11 is the resistant donor genotype,i.e. in one aspect at least one, optionally at least two, optionally allthree, or optionally all four of the following four SNPs on chromosome11 has the genotype of the resistant donor as indicated in the Tablebelow. As the introgression fragment on chromosome 11 needs to be inhomozygous form to confer the ToLCNDV resistance phenotype, the SNPgenotype is indicated in homozygous form.

In another aspect the SNP genotype of the melon plant comprising theintrogression fragment on chromosome 12 is the resistant donor genotype,i.e. in one aspect at least one, optionally at least two of the SNPs onchromosome 12, selected from SNP_05, SNP_06 and SNP_07, has/have thegenotype of the resistant donor as indicated in the Table 1 below.Optionally all three SNPs have the resistant donor genotype as indicatedin the Table 1 below. Although the introgression fragment on chromosome12 does not need to be in homozygous form to confer the ToLCNDVresistance phenotype, homozygous form is preferred. Therefore the SNPgenotype is indicated in homozygous form.

It is noted that SNP_01, SNP_02, SNP_03 and SNP_04 are physicallylocated on chromosome 11 in that order, as are SNP_05, SNP_06 and SNP_07on chromosome 12. An introgression fragment may therefore comprise thedonor SNP genotype for all four SNP markers linked to QTL11 and/or forall three SNP markers linked to QTL12 (as in the seeds depositedherein), or a smaller fragment, whereby one or more of the SNP markersis not present. As described further below, even all or all but onedonor SNP markers may be absent, while QTL11 or QTL12 is still presenton the introgression fragment.

TABLE 1 Resistant donor QTL on SNP genotype (in Chromo- SNP (SingleNucleotide Polymorphism) homozygous form) some SNP_01 refers tonucleotide 101 of SEQ ID NO: 1 (or of a sequence AA 11 comprising atleast 90%, or at least 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO: 1) SNP_02 refers to nucleotide 101 of SEQ ID NO: 2 (or of asequence GG 11 comprising at least 90%, or at least 95%, 96%, 97%, 98%or 99% sequence identity to SEQ ID NO: 2) SNP_03 refers nucleotide 101of SEQ ID NO: 3 (or of a sequence TT 11 comprising at least 90%, or atleast 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3)SNP_04 refers nucleotide 101 of SEQ ID NO: 4 (or of a sequence TT 11comprising at least 90%, or at least 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 4) SNP_05 refers nucleotide 101 of SEQ ID NO: 5(or of a sequence CC 12 comprising at least 90%, or at least 95%, 96%,97%, 98% or 99% sequence identity to SEQ ID NO: 5) SNP_06 refersnucleotide 101 of SEQ ID NO: 6 (or of a sequence GG 12 comprising atleast 90%, or at least 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO: 6) SNP_07 refers nucleotide 101 of SEQ ID NO: 7 (or of asequence TT 12 comprising at least 90%, or at least 95%, 96%, 97%, 98%or 99% sequence identity to SEQ ID NO: 7)

The nucleotide sequences (SEQ ID NO: 1 to SEQ ID NO: 7) comprising theSNPs provided herein are the nucleotide sequences of the resistantdonor, i.e. they contain the donor SNP nucleotide. Therefore, in oneaspect the present invention relates to cultivated melon plant cells ormelon plants (or plant parts) comprising an introgression fragment fromchromosome 11 and/or 12 of a ToLCNDV resistant donor plant, wherein theintrogression fragment confers ToLCNDV resistance and the introgressionfragment is detectable by (comprises) SEQ ID NO: 1 or an Adenine atnucleotide 101 of SEQ ID NO: 1 or an Adenine at the equivalentnucleotide of a sequence having substantial sequence identity to SEQ IDNO: 1 and/or by SEQ ID NO: 2 or a Guanine at nucleotide 101 of SEQ IDNO: 2 or a Guanine at the equivalent nucleotide of a sequence havingsubstantial sequence identity to SEQ ID NO:2 and/or by SEQ ID NO: 3 or aThymine at nucleotide 101 of SEQ ID NO: 3 or a Thymine at the equivalentnucleotide of a sequence having substantial sequence identity to SEQ IDNO: 3 and/or by SEQ ID NO: 4 or a Thymine at nucleotide 101 of SEQ IDNO: 4 or a Thymine at the equivalent nucleotide of a sequence havingsubstantial sequence identity to SEQ ID NO: 4 for the fragment onchromosome 11; and/or wherein the introgression fragment confers ToLCNDVresistance and the introgression fragment is detectable by (comprises)SEQ ID NO: 5 or a Cytosine at nucleotide 101 of SEQ ID NO: 5 or aCytosine at the equivalent nucleotide of a sequence having substantialsequence identity to SEQ ID NO:5 and/or by SEQ ID NO: 6 or a Guanine atnucleotide 101 of SEQ ID NO: 6 or a Guanine at the equivalent nucleotideof a sequence having substantial sequence identity to SEQ ID NO: 6and/or by SEQ ID NO: 7 or a Thymine at nucleotide 101 of SEQ ID NO: 7 ora Thymine at the equivalent nucleotide of a sequence having substantialsequence identity to SEQ ID NO: 7 for the fragment on chromosome 12. TheToLCNDV resistance conferring QTL is present on the introgressionfragment.

In one aspect only the QTL on chromosome 11 or only the QTL onchromosome 12 is introgressed into cultivated melon. The QTL onchromosome 11 has a better effect on ToLCNDV resistance (when inhomozygous form) than the QTL on chromosome 12. The QTL on chromosome 11confers an average ToLCNDV resistance score of at least 7.0 to thecultivated melon, compared to an average score of 4.0 or less, or 3.0 orless of a control line or variety lacking the QTL, such as a ToLCNDVsusceptible variety.

The QTL on chromosome 12, on the other hand, also confers ToLCNDVresistance when it is in heterozygous form. The QTL on chromosome 12confers an average ToLCNDV resistance score of at least 5.0 or at least6.0 to the cultivated melon, compared to a score of 4.0 or less, or 3.0or less of a control line or variety lacking the QTL, such as a ToLCNDVsusceptible variety.

In another aspect both QTLs, the QTL on chromosome 11 and the QTL onchromosome 12, are introgressed into cultivated melon. In one aspect atleast the QTL on chromosome 11 is in homozygous form. In another aspect,both QTLs are in homozygous form. The presence of both QTLs, with bothQTLs in homozygous form, provides the highest average resistance score,of at least 8.0, preferably a score of 9.0 (no symptoms).

The donor has an average score of 9.0 (no symptoms). Off course otherdonors, which have an average score of 9.0 may also contain QTL11 and/orQTL12. The SNP markers provided herein can be used to screen such donorsfor the presence of QTL 11 and QTL12.

In another aspect, one or more, or even all, or all but one of the SNPslinked to the QTL may be from the recipient, e.g. the susceptible melonplant, while the region between the SNPs is from the resistant donor andconfers ToLCNDV resistance, i.e. the resistance conferring donorfragment lies in-between the SNP markers. For example, a plant maycomprise the introgression fragment comprising the sequence of theToLCNDV resistant donor melon plant (carrying QTL11) in-between SNP_03and SNP_04, this plant in one aspect comprises a Cytosine (C) atnucleotide 101 of SEQ ID NO: 3 and a Cytosine (C) at nucleotide 101 ofSEQ ID NO: 4, i.e. the recipient nucleotides, not the donor. Thus, inone aspect only a region (the whole region or a part thereof) betweenthese two SNPs is from the donor, while SNP_03 and SNP_04 are from therecipient, having a Cytosine (C) at nucleotide 101 of SEQ ID NO: 3 and aCytosine (C) at nucleotide 101 of SEQ ID NO: 4. The same holds for otherpairs of SNPs.

In one aspect, at least SNP_04 in the cultivated melon plant, plant partor plant cell of the invention is from the resistant donor and has thedonor genotype, i.e. the introgression fragment comprises QTL11 and SEQID NO: 4.

In another aspect, at least SNP_06 in the cultivated melon plant, plantpart or plant cell of the invention is from the resistant donor and hasthe donor genotype, i.e. the introgression fragment comprises QTL12 andSEQ ID NO: 6.

In a preferred embodiment of the invention the introgression fragmentfrom chromosome 11 of the donor plant comprising the sequence of thedonor plant (comprising QTL11) in-between SNP_01 and SNP04, orin-between SNP_01 and SNP_03, or in-between SNP_01 and SNP_02, orbetween SNP_02 and SNP_04, or in-between SNP_02 and SNP_03, orin-between SNP_03 and SNP_04, or preferably in-between SNP_02 and SNP_04confers resistance to ToLCNDV to the melon plant cells according to theinvention or to the melon plants according to the invention.

In a preferred embodiment of the invention the introgression fragmentfrom chromosome 12 of the donor plant comprising the sequence of thedonor plant (comprising QTL12) in-between SNP_05 and SNP_07, or inbetween SNP_05 and SNP_06, or in-between SNP_06 and SNP_07 confersresistance to ToLCNDV to the melon plant cells according to theinvention or to the melon plants according to the invention.

Preferably, the melon plant cell according to the invention originatesfrom a cultivated melon plant or the melon plant according to theinvention, is a cultivated melon plant.

“Donor plant cell” or “donor plant” in connection with the presentinvention shall mean a melon plant cell or melon plant being resistantto ToLCNDV, preferably having an average resistance score of at least9.0. Likewise, the term DNA fragment or introgression fragment from thedonor plant or cell shall mean a fragment of chromosome 11 or 12 of amelon plant resistant to ToLCNDV, whereby the fragment confers ToLCNDVresistance when transferred into a ToLCNDV susceptible melon plant. Inone embodiment of the invention, the donor plant is a wild species orwild accession of melon. In one embodiment of the invention, DNAfragments or introgression fragments from donor plant cells or plantsare the donor fragments obtained from, or obtainable from, or as presentin, plants grown from seeds deposited under NCIMB 42625 or progenyobtained from plants grown from seeds deposited under NCIMB 42625 orplants obtained by crosses with plants grown from seeds deposited underNCIMB 42625. As mentioned, the deposited seeds are of cultivated melon(Piel de Sapo), into which QTL 11 and QTL12 has been introgressed from adonor plant which had an average disease score of 9.0.

Donor melon plants can be obtained from various sources. A personskilled in the art knows how to detect other sources of ToLCNDVresistant donor plants. For detecting such sources of ToLCNDV resistantdonor plants, basically melon plants can be infected with ToLCNDV,either by mechanical means, as described in Lopez et al. (2015,Euphytica 204(3), 679-691) or by transmission of the virus bywhiteflies. Preferably, infection occurs by whitefly infection incontext with the present invention. Plants showing reduced symptomlevels compared to susceptible controls can then be selected and used assource for genome fragments or sequences conferring ToLCNDV resistance.A preferred method on how to infect melon plants with ToLCNDV andmethods for determining the symptom level of infected plants are givenherein under “General Methods”.

In context of the present invention the donor plants preferably have anaverage symptom level equal to or above 8.0 and most preferred equal toor above 9.0. In one aspect the donor plant comprises the same SNPgenotype as shown in Table 3 for the donor for SNP_01, SNP_02, SNP_03and SNP_04. In one aspect the donor plant comprises the same SNPgenotype as shown in Table 3 for the donor for SNP_01, SNP_02, SNP_03and SNP_04 and for SNP_05, SNP_06, and optionally also for SNP_07.Preferably the SNP donor genotype is homozygous.

“Recurrent plant cell” or “recurrent plant” or “recipient plant” inconnection with the present invention shall be understood to be a melonplant cell or melon plant being sensitive (used herein synonymously withsusceptible) or non-resistant to ToLCNDV infection. If a plant issensitive or non-resistant to ToLCNDV can be determined by observationof the symptom levels after ToLCNDV infection. A recurrent plantpreferably has an average symptom level equal to or below 4.0, equal toor below 3.0, more preferably equal to or below 2.0. Symptom levels andmethods how to infect melon plants with ToLCNDV are described elsewhereherein and are applicable here accordingly. In a preferred embodiment ofthe invention, the recurrent melon plant cell according to the inventionoriginates from a cultivated melon plant or the recurrent melon plantaccording to the invention, is a cultivated melon plant. Preferably itis an elite line, breeding line or variety.

“Introgression fragment” refers to a chromosome fragment, chromosomepart or region which has been introduced into another plant of the sameor related species by crossing or traditional breeding techniques. Theintrogression of the fragment from a donor plant into a recurrent plantintroduces into the offspring of a cross between the donor and recurrentplant a phenotype, which was not present in the recurrent plant.Concerning the present invention, the phenotype transferred from thedonor plant to the recurrent plant is resistance to ToLCNDV, especiallyan average disease score of 7.0 or more, 8.0 or more or 9.0 or more. Forintrogression of a fragment into a specific elite line or variety thefirst crossing step can e.g. be followed by one or more backcrossingswith the intended elite line or variety. As understood herein,introgression can mean a first crossing of a ToLCNDV resistant donorplant with a ToLCNDV non-resistant recurrent plant and furtherback-crossing one or several times ToLCNDV resistant plants obtainedfrom the crossing with plants of the recipient into which ToLCNDVresistance shall be introgressed. In such a case, the introgressedfragment is the result of breeding methods referred to by the verb “tointrogress” (such as backcrossing) into a recipient variety or breedingline or elite line. Thus, introgression of ToLCNDV resistance into arecurrent plant is a technical process directed by man. In particularintrogression herein refers to a man-made breeding process or method.One or more or all of the molecular markers provided herein can be usedin that process. The resulting plant, i.e. the cultivated line orvariety comprising one or two introgression fragments from a donor isalso man-made and does not exist in nature.

The introgression fragment can be large, e.g. even half of a chromosome,but is preferably smaller, such as about 15 Mb (Megabases) or less, suchas about 10 Mb or less, about 9 Mb or less, about 8 Mb or less, about 7Mb or less, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less,about 3.5 or 3 Mb or less, about 2 Mb or less, about 1 Mb (equals1,000,000 base or less), or about 0.9 Mb (equals 900,000 base pairs) orless, such as 0.8 Mb, 0.7 Mb, 0.6 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, or less.

The physical distance between SNP_01 and SNP_04 is about 1.16 Mb, andthe physical distance between SNP_05 and SNP_06 is about 0.88 Mb andbetween SNP_05 and SNP_07 about 3.4 Mb. So in one aspect theintrogression fragment comprises the elsewhere herein indicated physicalregion comprising one or more of the SNPs, or a sub-region thereof,which retains the QTL.

The introgression fragment can originate from e.g. a wild melon plant orwild melon accession or wild relatives of melon or landraces (donor).Wild melon plants or wild melon accessions or wild relatives of melonplants or landraces can be used to introgress fragments of the donorgenome into the genome of cultivated melon, Cucumis melo, to generateelite lines and varieties with good agronomic characteristics. Such acultivated melon plant thus has a “genome of cultivated C. melo”, butcomprises in its genome a fragment of the donor, e.g. an introgressionfragment of a related Cucumis genome, such as Cucumis melo ssp.agrestis, C. melo ssp. melo, C. melo ssp. acidulous, C. callosus, C.trigonus, C. picrocarpus, or another wild melon or wild relative ofmelon. It is understood that the term “introgression fragment” neverincludes a whole chromosome, but only a part of a chromosome. Thechromosomes carrying the introgression therefore also comprise a part orparts of the recurrent (recipient) melon plant and in addition parts ofthe donor melon plant.

In one aspect the donor plant of the invention is not one of the fiveCucumis melo subsp. agrestis accessions (subsp. agrestis var. momordica:Mom-KhaInd/Kharbuja, Mom-PI124Ind/PI124112, Mom-PI124Ind/PI414723 andsubsp. agrestis wild types: Ag-WM9Ind/WM9, Ag-WM7Ind/WM7) resistant toToLCNDV identified by Lopez et al., 2015, Euphytica 204(3), 679-691.

In another aspect the donor plant of the invention is not an accessionbelonging to the subspecies (abbreviated as ssp′ or ‘subsp.’) agrestis,i.e. it is not a Cucumis melo ssp agrestis accession, especially in oneaspect the donor is not a Cucumis melo ssp agrestis var. acidulousaccession. In one aspect the resistance donor is a Cucumis melo ssp.melo accession.

The term “breeding” encompasses herein crossing, backcrossing, selfing,selection, double haploid production, embryo rescue, protoplast fusion,marker assisted selection, mutation breeding etc. as known to thebreeder (i.e. methods other than geneticmodification/transformation/transgenic methods), by which, for example,a recombinant chromosome 11 or 12 can be obtained, identified, producedand/or transferred.

In a preferred embodiment of the present invention, the introgressionfragment originates form a wild Cucumis plant or a wild Cucumisaccession or a landrace, most preferably the introgression fragmentoriginates from a Cucumis melo ssp. melo, having one or more of thefollowing characteristics: a low brix or a low percentage of TSS (totalsoluble solids, e.g. less than 6% Total Soluble Solids or less than 5.5%TSS), a very weak attachment of the peduncle at fruit maturity (on ascale of very weak, weak, medium, strong, very strong, see UPOVcharacteristic 39 in TG/104/5 Rev., UPOV Code: CUCUM_MEL, found on thewebsite UPOV.int), a very short shelf life of the fruit (on a scale ofvery short, short, medium, long, very long; see UPOV characteristic 68in TG/104/5 Rev., UPOV Code: CUCUM_MEL, found on the website UPOV.int).

“Plant variety” is a group of plants within the same botanical taxon ofthe lowest grade known, which (irrespective of whether the conditionsfor the recognition of plant breeder's rights are fulfilled or not) canbe defined on the basis of the expression of characteristics that resultfrom a certain genotype or a combination of genotypes, can bedistinguished from any other group of plants by the expression of atleast one of those characteristics, and can be regarded as an entity,because it can be multiplied without any change. Therefore, the term“plant variety” cannot be used to denote a group of plants, even if theyare of the same kind, if they are all characterized by the presence ofone or two loci or genes (or phenotypic characteristics due to thesespecific loci or genes), but which can otherwise differ from one anotherenormously as regards the other loci or genes.

“F1, F2, F3, etc.” refers to the consecutive related generationsfollowing a cross between two parent plants or parent lines. The plantsgrown from the seeds produced by crossing two plants or lines is calledthe F1 generation. Selfing the F1 plants results in the F2 generation,etc.

“F1 hybrid” plant (or F1 hybrid seed) is the generation obtained fromcrossing two inbred parent lines. Thus, F1 hybrid seeds are seeds fromwhich F1 hybrid plants grow. F1 hybrids are more vigorous and higheryielding, due to heterosis. Inbred lines are essentially homozygous atmost loci in the genome.

A “plant line” or “breeding line” refers to a plant and its progeny. Asused herein, the term “inbred line” refers to a plant line which hasbeen repeatedly selfed and is nearly homozygous. Thus, an “inbred line”or “parent line” refers to a plant which has undergone severalgenerations (e.g. at least 5, 6, 7 or more) of inbreeding, resulting ina plant line with a high uniformity.

“Uniformity” or “uniform” relates to the genetic and phenotypiccharacteristics of a plant line or variety. Inbred lines are geneticallyhighly uniform as they are produced by several generations ofinbreeding. Likewise, and the F1 hybrids which are produced from suchinbred lines are highly uniform in their genotypic and phenotypiccharacteristics and performance.

“Chromosome 11 of a melon plant” is to be understood in context of thepresent invention as the scaffolds, fragments, regions, markers andnucleic acid sequences assigned by the ICuGI (International CucurbitGenomics Initiative) to belong to chromosome 11 of the melon genome orpreferably by melonomics.net assigned to chromosome 11. Onmelonomics.net one can BLAST sequences against the melon genomescaffolds under Old Melonomics (v3.5) or against the melon genomeversion CM3.5.1 or CM3.6.1 (New Melonomics v4.0).

“Orthologous chromosome 11” refers to the chromosome 11 of donor plants,parts of which can be introgressed into cultivated melon chromosome 11.

“Chromosome 12 of a melon plant” is to be understood in context of thepresent invention as the scaffolds, fragments, regions, markers andnucleic acid sequences assigned by the ICuGI (International CucurbitGenomics Initiative) to belong to chromosome 12 of the melon genome orpreferably by melonomics.net assigned to chromosome 12.

“Orthologous chromosome 12” refers to the chromosome 12 of donor plants,parts of which can be introgressed into cultivated melon chromosome 12.

“ICuGI” refers herein to the Cucumis melo data published by theInternational Cucurbit Genomics Initiative, which publishes genetic mapsof e.g. Cucumis melo (http://www.icugi.org/cgi-bin/cmap/mapset_info?species acc=CM), but which was renewed in May 2017 and can befound at cucurbitgenomics.org. Further information including additionalmarkers and mapping information in addition to the ICuCI data isavailable from Diaz et al. (2015, Mol Breeding 35, 188) and theadditional data included in the online version of the respectivearticle.

Scaffold CM3.5_scaffold00052 of melonomics.net corresponds to chromosome11 (under Old Melonomics v3.5). Scaffold CM3.5_scaffold00004 ofmelonomics.net corresponds to chromosome 12 (under Old Melonomics v3.5).If one BLASTs any of the sequences of SEQ ID NO: 1 to SEQ ID NO: 7provided herein against the published sequence of the melon genome ofthe melonomics.net database (under Old Melonomics v3.5), the alignmentwith the scaffold will be shown. If one BLASTs any one of SEQ ID NO: 1to SEQ ID NO: 7 provided herein against the CM3.6.1 genome or theCM3.5.1 genome (under New Melonomics v4.0), the chromosome and alignmentwith the chromosome part of the genome will be shown.

“Cultivated melon plant” refers to plants of Cucumis melo i.e.varieties, breeding lines or cultivars of the species C. melo,cultivated by humans and having good agronomic characteristics,especially producing edible and marketable fruits of good size andquality and uniformity; preferably such plants are not “wild melonplants”, i.e. plants which generally have much poorer yields and pooreragronomic characteristics than cultivated plants and e.g. grow naturallyin wild populations. “Wild melon plants” include for example ecotypes,PI (Plant Introduction) lines, landraces or wild accessions or wildrelatives of a species.

A “SNP (=Single Nucleotide Polymorphism)” in context with the presentinvention is to be understood as a variation in a single nucleotide thatoccurs at a specific position in the genome. A SNP is the variation ofthe single nucleotide at the given position in a genome between twoplants. If a wild melon plant having a ToLCNDV resistance (donor plant)shows in its corresponding sequence at a specific single position anucleotide which is different from the corresponding nucleotide at thesame position of a cultivated melon plant, the position defines a SNPbetween the wild melon and the cultivated melon. If the donor plant hasone of the four possible nucleotides (A, C, T or G) at a specificposition, a SNP occurs, when the cultivated plant has either of theremaining three possible nucleotides at the same corresponding sequenceposition. In a cultivated melon plant comprising an introgressionfragment from a donor, it can therefore easily be determined if thesingle nucleotide of the SNP is from the donor or from the cultivatedmelon (recipient).

“SNP_01” which is alternatively designated “mME72223” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO: 1. SEQ ID NO:1 or a sequence substantially identicalto SEQ ID NO:1 can be found on chromosome 11 in e.g. the ICuGI data setor on melonomics.net. The relative position of SNP_01 on the physicalchromosome can be found in Table 2. Also the position on the genomeversion CM3.6.1 (New Melonomics v4.0) is shown. Preferably thenucleotide sequence comprising SNP_01 has a nucleotide sequence havingat least 85% sequence identity with the nucleotide sequence shown underSEQ ID NO: 1, more preferably at least 90% identity with the nucleotidesequence shown under SEQ ID NO: 1, further more preferably at least 95%identity with the nucleotide sequence shown under SEQ ID NO: 1, evenmore preferred at least 97% identity with the nucleotide sequence shownunder SEQ ID NO: 1, even further more preferred at least 98% identitywith the nucleotide sequence shown under SEQ ID NO: 1, in particularpreferred at least 99% identity with the nucleotide sequence shown underSEQ ID NO: 1 under the provision that in each case the nucleotide atposition 101 in SEQ ID NO: 1 is different from the correspondingnucleotide at the same position of the recurrent plant. Such sequenceshaving at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identityto SEQ ID NO: 1 are referred to as having substantial sequence identityto SEQ ID NO: 1.

The ToLCNDV resistant donor plant used in the invention has an ‘A’(Adenine) at position 101 of SEQ ID NO: 1. In a preferred embodiment ofthe invention, SNP_01 is characterized in that the recurrent plant hasan C, G or T at position 101 in SEQ ID NO: 1. In one aspect therecurrent plant has a G at position 101 in SEQ ID NO: 1. In oneembodiment of the invention, SNP_01 is characterized in that the ToLCNDVresistant donor plant, or introgression fragment derived from the donorplant, has a ‘A’ at position 101 in SEQ ID NO: 1 and the recurrent planthas a G at position 101 in SEQ ID NO: 1, or a C at position 101 or a Tat position 101 of SEQ ID NO: 1 (or at the equivalent position of asequence comprising substantial sequence identity to SEQ ID NO: 1).

“SNP_02” which is alternatively designated “mME72233” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO: 2. SEQ ID NO:2 or a sequence substantially identicalto SEQ ID NO:2 can be found on chromosome 11 e.g. in the ICuGI data setor on melonomics.net. The relative position of SNP_02 on the physicalchromosome can be found in Table 2. Also the position on the genomeversion CM3.6.1 (New Melonomics v4.0) is shown. Preferably thenucleotide sequence comprising SNP_02 has a nucleotide sequence havingat least 85% sequence identity with the nucleotide sequence shown underSEQ ID NO: 2, more preferably at least 90% identity with the nucleotidesequence shown under SEQ ID NO: 2, further more preferably at least 95%identity with the nucleotide sequence shown under SEQ ID NO: 2, evenmore preferred at least 97% identity with the nucleotide sequence shownunder SEQ ID NO: 2, even further more preferred at least 98% identitywith the nucleotide sequence shown under SEQ ID NO: 2, in particularpreferred at least 99% identity with the nucleotide sequence shown underSEQ ID NO: 2 under the provision that in each case the nucleotide atposition 101 in SEQ ID NO: 2 is different from the correspondingnucleotide at the same position of the recurrent plant. Such sequenceshaving at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identityto SEQ ID NO: 2 are referred to as having substantial sequence identityto SEQ ID NO: 2.

The ToLCNDV resistant donor plant used in the invention has an ‘G’(Guanine) at position 101 of SEQ ID NO: 2. In a preferred embodiment ofthe invention, SNP_02 is characterized in that the recurrent plant has aC, A or T at position 101 in SEQ ID NO: 2. In one aspect the recurrentplant has an ‘A’ at position 101 in SEQ ID NO: 2. In one embodiment ofthe invention, SNP_02 is characterized in that the ToLCNDV resistantdonor plant, or introgression fragment derived from the donor plant, hasa ‘G’ at position 101 in SEQ ID NO: 2 and the recurrent plant has an ‘A’at position 101 in SEQ ID NO: 2, or a C at position 101 or a T atposition 101 of SEQ ID NO: 2 (or at the equivalent position of asequence comprising substantial sequence identity to SEQ ID NO: 2).

“SNP_03” which is alternatively designated “mME72238” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO: 3. SEQ ID NO:3 or a sequence substantially identicalto SEQ ID NO:3 can be found on chromosome 11 e.g. in the ICuGI data setor on melonomics.net. The relative position of SNP_03 on the physicalchromosome can be found in Table 2. Also the position on the genomeversion CM3.6.1 (New Melonomics v4.0) is shown. Preferably thenucleotide sequence comprising SNP_03 has a nucleotide sequence havingat least 85% sequence identity with the nucleotide sequence shown underSEQ ID NO: 3, more preferably at least 90% identity with the nucleotidesequence shown under SEQ ID NO: 3, further more preferably at least 95%identity with the nucleotide sequence shown under SEQ ID NO: 3, evenmore preferred at least 97% identity with the nucleotide sequence shownunder SEQ ID NO: 3, even further more preferred at least 98% identitywith the nucleotide sequence shown under SEQ ID NO: 3, in particularpreferred at least 99% identity with the nucleotide sequence shown underSEQ ID NO: 3 under the provision that in each case the nucleotide atposition 101 in SEQ ID NO: 3 is different from the correspondingnucleotide at the same position of the recurrent plant. Such sequenceshaving at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identityto SEQ ID NO: 3 are referred to as having substantial sequence identityto SEQ ID NO: 3.

The ToLCNDV resistant donor plant used in the invention has an ‘T’(Thymine) at position 101 of SEQ ID NO: 3. In a preferred embodiment ofthe invention, SNP_03 is characterized in that the recurrent plant hasan C, A or G at position 101 in SEQ ID NO: 3. In one aspect therecurrent plant has a C at position 101 in SEQ ID NO: 3. In oneembodiment of the invention, SNP_03 is characterized in that the ToLCNDVresistant donor plant, or introgression fragment derived from the donorplant, has a ‘T’ at position 101 in SEQ ID NO: 3 and the recurrent planthas a C at position 101 in SEQ ID NO: 3, or a A at position 101 or a Gat position 101 of SEQ ID NO: 3 (or at the equivalent position of asequence comprising substantial sequence identity to SEQ ID NO: 3).

“SNP_04” which is alternatively designated “mME72245” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO: 4. SEQ ID NO:4 or a sequence substantially identicalto SEQ ID NO:4 can be found on chromosome 11 e.g. in the ICuGI data setor on melonomics.net. The relative position of SNP_04 on the physicalchromosome can be found in Table 2.

Also the position on the genome version CM3.6.1 (New Melonomics v4.0) isshown. Preferably the nucleotide sequence comprising SNP_04 has anucleotide sequence having at least 85% sequence identity with thenucleotide sequence shown under SEQ ID NO: 4, more preferably at least90% identity with the nucleotide sequence shown under SEQ ID NO: 4,further more preferably at least 95% identity with the nucleotidesequence shown under SEQ ID NO: 4, even more preferred at least 97%identity with the nucleotide sequence shown under SEQ ID NO: 4, evenfurther more preferred at least 98% identity with the nucleotidesequence shown under SEQ ID NO: 4, in particular preferred at least 99%identity with the nucleotide sequence shown under SEQ ID NO: 4 under theprovision that in each case the nucleotide at position 101 in SEQ ID NO:4 is different from the corresponding nucleotide at the same position ofthe recurrent plant. Such sequences having at least 85%, 90%, 95%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 4 are referred to ashaving substantial sequence identity to SEQ ID NO: 4.

The ToLCNDV resistant donor plant used in the invention has an ‘T’(Thymine) at position 101 of SEQ ID NO: 4. In a preferred embodiment ofthe invention, SNP_04 is characterized in that the recurrent plant hasan C, A or G at position 101 in SEQ ID NO: 4. In one aspect therecurrent plant has a C at position 101 in SEQ ID NO: 4. In oneembodiment of the invention, SNP_04 is characterized in that the ToLCNDVresistant donor plant, or introgression fragment derived from the donorplant, has a ‘T’ at position 101 in SEQ ID NO: 4 and the recurrent planthas a C at position 101 in SEQ ID NO: 4, or a A at position 101 or a Gat position 101 of SEQ ID NO: 4 (or at the equivalent position of asequence comprising substantial sequence identity to SEQ ID NO: 4).

“SNP_05” which is alternatively designated “mME72255” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO: 5. SEQ ID NO:5 or a sequence substantially identicalto SEQ ID NO:5 can be found on chromosome 12 e.g. in the ICuGI data setor on melonomics.net. The relative position of SNP_05 on the physicalchromosome can be found in Table 2. Also the position on the genomeversion CM3.6.1 (New Melonomics v4.0) is shown. Preferably thenucleotide sequence comprising SNP_05 has a nucleotide sequence havingat least 85% sequence identity with the nucleotide sequence shown underSEQ ID NO: 5, more preferably at least 90% identity with the nucleotidesequence shown under SEQ ID NO: 5, further more preferably at least 95%identity with the nucleotide sequence shown under SEQ ID NO: 5, evenmore preferred at least 97% identity with the nucleotide sequence shownunder SEQ ID NO: 5, even further more preferred at least 98% identitywith the nucleotide sequence shown under SEQ ID NO: 5, in particularpreferred at least 99% identity with the nucleotide sequence shown underSEQ ID NO: 5 under the provision that in each case the nucleotide atposition 101 in SEQ ID NO: 5 is different from the correspondingnucleotide at the same position of the recurrent plant. Such sequenceshaving at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identityto SEQ ID NO: 5 are referred to as having substantial sequence identityto SEQ ID NO: 5.

The ToLCNDV resistant donor plant used in the invention has an ‘C’(Cytosine) at position 101 of SEQ ID NO: 5. In a preferred embodiment ofthe invention, SNP_05 is characterized in that the recurrent plant hasan G, A or T at position 101 in SEQ ID NO: 5. In one aspect therecurrent plant has a T at position 101 in SEQ ID NO: 5. In oneembodiment of the invention, SNP_05 is characterized in that the ToLCNDVresistant donor plant, or introgression fragment derived from the donorplant, has a ‘C’ at position 101 in SEQ ID NO: 5 and the recurrent planthas a Tat position 101 in SEQ ID NO: 5, or a G at position 101 or an Aat position 101 of SEQ ID NO: 5 (or at the equivalent position of asequence comprising substantial sequence identity to SEQ ID NO: 5).

“SNP_06” which is alternatively designated “mME72261” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO: 6. SEQ ID NO:6 or a sequence substantially identicalto SEQ ID NO:6 can be found on chromosome 12 e.g. in the ICuGI data setor on melonomics.net. The relative position of SNP_06 on the physicalchromosome can be found in Table 2. Also the position on the genomeversion CM3.6.1 (New Melonomics v4.0) is shown. Preferably thenucleotide sequence comprising SNP_06 has a nucleotide sequence havingat least 85% sequence identity with the nucleotide sequence shown underSEQ ID NO: 6, more preferably at least 90% identity with the nucleotidesequence shown under SEQ ID NO: 6, further more preferably at least 95%identity with the nucleotide sequence shown under SEQ ID NO: 6, evenmore preferred at least 97% identity with the nucleotide sequence shownunder SEQ ID NO: 6, even further more preferred at least 98% identitywith the nucleotide sequence shown under SEQ ID NO: 6, in particularpreferred at least 99% identity with the nucleotide sequence shown underSEQ ID NO: 6 under the provision that in each case the nucleotide atposition 101 in SEQ ID NO: 6 is different from the correspondingnucleotide at the same position of the recurrent plant. Such sequenceshaving at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identityto SEQ ID NO: 6 are referred to as having substantial sequence identityto SEQ ID NO: 6.

The ToLCNDV resistant donor plant used in the invention has an ‘G’(Guanine) at position 101 of SEQ ID NO: 6. In a preferred embodiment ofthe invention, SNP_06 is characterized in that the recurrent plant hasan C, A or T at position 101 in SEQ ID NO: 6. In one aspect therecurrent plant has a A at position 101 in SEQ ID NO: 6. In oneembodiment of the invention, SNP_06 is characterized in that the ToLCNDVresistant donor plant, or introgression fragment derived from the donorplant, has a ‘G’ at position 101 in SEQ ID NO: 6 and the recurrent planthas an A at position 101 in SEQ ID NO: 6, or a C at position 101 or a Tat position 101 of SEQ ID NO: 6 (or at the equivalent position of asequence comprising substantial sequence identity to SEQ ID NO: 6).

“SNP_07” which is alternatively designated “mME72279” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO: 7. SEQ ID NO:7 or a sequence substantially identicalto SEQ ID NO:7 can be found on chromosome 12 e.g. in the ICuGI data setor on melonomics.net. The relative position of SNP_07 on the physicalchromosome can be found in Table 2. Also the position on the genomeversion CM3.6.1 (New Melonomics v4.0) is shown. Preferably thenucleotide sequence comprising SNP_07 has a nucleotide sequence havingat least 85% sequence identity with the nucleotide sequence shown underSEQ ID NO: 7, more preferably at least 90% identity with the nucleotidesequence shown under SEQ ID NO: 7, further more preferably at least 95%identity with the nucleotide sequence shown under SEQ ID NO: 7, evenmore preferred at least 97% identity with the nucleotide sequence shownunder SEQ ID NO: 7, even further more preferred at least 98% identitywith the nucleotide sequence shown under SEQ ID NO: 7, in particularpreferred at least 99% identity with the nucleotide sequence shown underSEQ ID NO: 7 under the provision that in each case the nucleotide atposition 101 in SEQ ID NO: 7 is different from the correspondingnucleotide at the same position of the recurrent plant. Such sequenceshaving at least 85%, 90%, 95%, 97%, 98%, 99% or more sequence identityto SEQ ID NO: 7 are referred to as having substantial sequence identityto SEQ ID NO: 7.

The ToLCNDV resistant donor plant used in the invention has an ‘T’(Thymine) at position 101 of SEQ ID NO: 7. In a preferred embodiment ofthe invention, SNP_07 is characterized in that the recurrent plant hasan C, A or G at position 101 in SEQ ID NO: 7. In one aspect therecurrent plant has a C at position 101 in SEQ ID NO: 7. In oneembodiment of the invention, SNP_07 is characterized in that the ToLCNDVresistant donor plant, or introgression fragment derived from the donorplant, has a ‘T’ at position 101 in SEQ ID NO: 7 and the recurrent planthas a C at position 101 in SEQ ID NO: 7, or a G at position 101 or a Aat position 101 of SEQ ID NO: 7 (or at the equivalent position of asequence comprising substantial sequence identity to SEQ ID NO: 7).

TABLE 2 Physical SNP nucleotide position position on melon SNP on thegenome version Scaffold (world nucleotide chromosome 11 CM3.6.1 (Newwide web at position on or 12 according Melonomics v4.0) melonomics.netscaffold00052 to Diaz et al. on chromosome 12 Chromo- Old Melonomics or2015* or chromosome 12, some Marker name v3.5) scaffold0004 e.g. FIG 1respectively 11 CMPSNP475 Scaffold00052 1308124 30277435 11 SNP_01(mME72223) Scaffold00052 1256506 33240885 11 SNP_02 (mME72233)Scaffold00052  777406 32761709 11 SNP_03 (mME72238) Scaffold00052 513312 32497404 11 SNP_04 (mME72245) Scaffold00052   90616 32074519 11ECM192 Scaffold00052   32422 29001733 12 CMPSNP2002 Scaffold00004 255636 12534642 12 SNP_05 (mME72255) Scaffold00004 3413496  9379373 12SNP_06 (mME72261) Scaffold00004 4297467  8494993 12 SNP_07 (mME72279)Scaffold00004 6896770  5895390 12 CMPSNP310 Scaffold00004 7646165 5144113 *SNP nucleotide position based on position in Diaz et al. 2015,Mol Breeding 35:188, p 1-7.

As can be seen, SNP_01, SNP_02, SNP_03 and SNP_04 lie in betweenCMPSNP475 (at 30.27 Mb) and ECM192 (at 29.0 Mb) on the physicalchromosome 11, i.e. at the lower half of chromosome 11. On the genomeCM3.6.1 SNP_01, SNP_02, SNP_03 and SNP_04 lie in the region starting at32.07 Mb and ending at 33.24 Mb of chromosome 11.

SNP_05, SNP_06 and SNP_07 lie in between CMPSNP2002 (at 12.5 Mb) andCMPSNP310 (at 5.1 Mb) on the physical chromosome 12, i.e. on the upperhalf of chromosome 12. On the genome CM3.6.1 SNP_05, SNP_06 and SNP_07lie in the region starting at 5.89 Mb and ending at 9.37 Mb ofchromosome 12.

The molecular markers described herein may be detected according tostandard methods. For example SNP markers can be detected using aKASP-assay (see \Vww.kpbioscience.co.uk) or other assays. A KASP-assayhas been developed for SNPs described herein. Respective details aredisclosed in the Example section. Sequences used in the respectiveKASP-assays are given in the Sequence Listing. For developingKASP-assays for the SNPs two allele specific forward primers and oneallele specific reverse primer were designed according to common generalknowledge (see e.g. Allen et al. 2011, Plant Biotechnology J. 9, 1 086-1 099, especially p097-098 for KASP assay method).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “essentially similar” when they areoptimally aligned by for example the programs GAP or BESTFIT or theEmboss program “Needle” (using default parameters, see below) share atleast a certain minimal percentage of sequence identity defined furtherbelow). These programs use the Needleman and Wunsch global alignmentalgorithm to align two sequences over their entire length, maximizingthe number of matches and minimizing the number of gaps. Generally, thedefault parameters are used, with a gap creation penalty=10 and gapextension penalty=0.5 (both for nucleotide and protein alignments). Fornucleotides the default scoring matrix used is DNAFULL and for proteinsthe default scoring matrix is Blosum62 (Henikoff & Henikoff, 1 992, PNAS89, 1 09 1 5-1 09 1 9). Sequence alignments and scores for percentagesequence identity may for example be determined using computer programs,such as EMBOSS as available on the world wide web underebi.ac.uk/Tools/psa/emboss_needle/). Alternatively sequence similarityor identity may be determined by searching against databases such asFASTA, BLAST, etc., but hits are preferably retrieved and alignedpairwise to compare sequence identity. Two proteins or two proteindomains, or two nucleic acid sequences have “substantial sequenceidentity” if the percentage sequence identity is at least 85%, 90%, 95%,98% or 99% (as determined by Emboss “needle” using default parameters,i.e. gap creation penalty=10, gap extension penalty=0.5, using scoringmatrix DNAFULL for nucleic acids an Blosum62 for proteins). Whenreference is made to a nucleic acid sequence (e.g. DNA or genomic DNA)having “substantial sequence identity to” a reference sequence or havinga sequence identity of at least 80%, e.g. at least 85%, 90%, 95%, 98%,99%, 99.2%, 99.5%, 99.9% nucleic acid sequence identity to a referencesequence, in one embodiment said nucleotide sequence is consideredsubstantially identical to the given nucleotide sequence and can beidentified using stringent hybridization conditions. In anotherembodiment, the nucleic acid sequence comprises one or more mutationscompared to the given nucleotide sequence but still can be identifiedusing stringent hybridisation conditions.

Cultivated melon plants comprising a ToLCNDV resistance conferringfragment introgressed from the donor on chromosome 11 and/or 12 showreduced symptoms when infected with ToLCNDV, while susceptible controls(lacking the introgression fragments on chromosome 11 and 12) show theexpected severe symptoms in the same conditions.

In a preferred embodiment of the invention, plants according to theinvention, or plant cells according to the invention, are characterizedin that they upon infection with ToLCNDV show an average symptom level(or average disease score) of at least 7.0, more preferably at least 8.0or 9.0. As mentioned previously, QTL11 confers high levels of resistancewhen the QTL is in homozygous form. A ToLCNDV susceptible plant intowhich the introgression fragment comprising QTL11 is introduced by e.g.backcrossing and which is selfed to generate a homozygous introgressionfragment (comprising the donor genotype for one or more or all ofSNP_01, SNP_02, SNP_03 and SNP_04 in homozygous form) will result in aplant which is highly resistant to ToLCNDV infection, having an averagedisease score of at least 7.0, preferably at least 8.0 or preferably atleast 9.0. QTL12 confers a lower level of resistance than QTL11, but ina partially dominant manner. A TolCNDV susceptible plant into which theintrogression fragment comprising QTL12 is introduced by e.g.backcrossing and optionally selfing to generate a homozygousintrogression fragment (comprising the donor genotype for one or more orall of SNP_05, SNP_6 and SNP_07 in heterozygous or homozygous form),will result in a plant having a higher level of resistance, with anaverage disease score of at least 5.0, preferably at least 6.0.

Symptom levels following infection with ToLCNDV are assessed on a scalebetween 1 and 9, wherein 1 is defined to be the level with the mostsevere symptoms and 9 is defined as the highest resistance level, havebeen described herein above and are applicable here accordingly. Apreferred test for determining the symptom levels is given below under“General Methods”.

Resistance to ToLCNDV conferred by the introgression fragment onchromosome 11 is expressed in a recessive manner and, thus, can beobserved only when both chromosomes 11 comprise the introgressionfragment comprising QTL11 and the donor SNP genotype of one or more orall of the SNP markers provided herein (SNP_01, SNP_02, SNP_03 andSNP_04) and/or the introgression fragment comprising the sequence of thedonor plant in-between the SNP markers of chromosome 11 disclosedherein.

Resistance to ToLCNDV conferred by the introgression fragment onchromosome 12 is expressed in a dominant manner and, thus, can beobserved when one of the chromosomes 12 comprises the introgressionfragment comprising QTL12 and the donor SNP genotype of one or more orall of the SNP markers provided herein (SNP_05, SNP_06 and SNP_07)and/or the introgression fragment comprising the sequence of the donorplant in-between the SNP markers of chromosome 12 disclosed herein.

A plant comprising both QTL11 in homozygous form and QTL12 inheterozygous or in homozygous form will have an average ToLCNDV diseasescore of at least 8.0, preferably at least 9.0, i.e. be highlyresistant. Such a plant has the advantage that the resistance is basedon two different QTLs, meaning that resistance breakdown is unlikely tooccur.

Other specific embodiments of the present invention therefore relate tocultivated melon plants, plant parts and plant cell according to theinvention wherein the introgression fragment from chromosome 11 of aToLCNDV resistant donor plant comprising the sequence of the donor plantin-between SNP_01 and SNP_04, or in-between SNP O1 and SNP_03, orin-between SNP O1 and SNP_02, or in-between SNP_02 and SNP_04, orbetween SNP_02 and SNP_03, or in-between SNP_03 and SNP_04 is present inhomozygous state. The introgression fragment comprises QTL11, andtherefore confers high ToLCNDV resistance to the plant.

In another aspect the present invention relates to cultivated melonplants, plant parts and plant cell according to the invention whereinthe introgression fragment from chromosome 12 of a ToLCNDV resistantdonor plant comprising the sequence of the donor plant in-between SNP_05and SNP_06, or in-between SNP_06 and SNP_07, or in-between SNP_05 andSNP_07 is present in heterozygous or homozygous state. The introgressionfragment comprises QTL12, and therefore confers ToLCNDV resistance tothe plant.

In a further aspect, a cultivated melon plant, plant part or plant cellis provided which comprises both QTL11 (in homozygous form) and QTL12(in heterozygous or in homozygous form) in its genome, i.e. it comprisestwo introgression fragments as described above and elsewhere herein.Such a melon plant can for example be generated by crossing a plantcomprising QTL11 with a plant comprising QTL12 and selecting progenywhich comprise both introgression fragments and both QTLs. Also the seeddeposit provided herein comprises both QTLs in homozygous form (with theresistant donor genotype being present in homozygous form for SNP_01,SNP_02, SNP_03, SNP_04, SNP_05, SNP_06 and SNP_07) can be used totransfer both QTLs into any other cultivated melon line or variety bycrossing the plant with another melon plant and optionally by selectingprogeny plants comprising the resistant donor genotype for at least oneSNP marker selected from SNP_01, SNP_02, SNP_03 and SNP_04 and at leastone SNP marker selected from SNP_05, SNP_06 and SNP_07, preferably atleast one SNP marker selected from SNP_05 and SNP_06. Optionally progenycan be tested for ToLCNDV resistance as described. Preferably, but notnecessarily, QTL11 and QTL12 are both from the same donor plant.

Table 3 illustrates the SNP genotype of melon plants or melon plantcells comprising the donor SNPs in homozygous form or heterozygous form,as well as the recurrent parent SNP genotype, lacking the introgressionfragment.

SNP genotype in SNP genotype in SNP genotype melon plant melon plant ofthe recurrent comprising the donor comprising the parent, lacking SNPand nucleotide position fragment in donor fragment in the introgression(nt) in the sequence homozygous form heterozygous form fragment SNP_01AA AG GG (nt 101 of SEQ ID NO 1) SNP_02 GG AG AA (nt 101 of SEQ ID NO 2)SNP_03 TT TC CC (nt 101 of SEQ ID NO 3) SNP_04 TT TC CC (nt 101 of SEQID NO 4) SNP_05 CC TC TT (nt 101 of SEQ ID NO 5) SNP_06 GG AG AA (nt 101of SEQ ID NO 6) SNP_07 TT TC CC (nt 101 of SEQ ID NO 7)

In one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 11, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment lies in-between SNP_01 and SNP_04 and whereby theintrogression fragment comprises a Thymine (T) at nucleotide 101 of SEQID NO: 3 or at the equivalent nucleotide of a sequence comprisingsubstantial sequence identity to SEQ ID NO: 3. In a further aspect theintrogression fragment further comprises a Thymine (T) at nucleotide 101of SEQ ID NO: 4 or at the equivalent nucleotide of a sequence comprisingsubstantial sequence identity to SEQ ID NO: 4 and/or the introgressionfragment further comprises a Guanine (G) at nucleotide 101 of SEQ ID NO:2 or at the equivalent nucleotide of a sequence comprising substantialsequence identity to SEQ ID NO:2. Optionally the introgression fragmentfurther comprises the resistant donor nucleotide of SNP_01.

In one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 11, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment lies in-between SNP_01 and SNP_04 and whereby theintrogression fragment comprises a Thymine (T) at nucleotide 101 of SEQID NO: 3 or at the equivalent nucleotide of a sequence comprisingsubstantial sequence identity to SEQ ID NO: 3 and the introgressionfragment optionally further comprises the resistant donor nucleotide ofSNP_02 and/or SNP_01. Optionally the introgression fragment furthercomprises the donor SNP genotype for SNP_04.

In one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 11, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence between atleast SNP_02 and SNP_03, optionally including the donor SNP nucleotidefor one or both of these SNP markers. So optionally the introgressionfragment comprises SEQ ID NO: 2 and SEQ ID NO: 3 and the nucleotidesequence in-between SEQ ID NO: 2 and SEQ ID NO: 3 is also from theresistant donor. Thus, the introgression fragment comprises the entireregion from SEQ ID NO: 2 to SEQ ID NO: 3.

In one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 11, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence in-betweenat least SNP_04 and SNP_03, optionally including the donor SNPnucleotide for one or both of these SNP markers. So optionally theintrogression fragment comprises SEQ ID NO: 4 and SEQ ID NO: 3 and thenucleotide sequence in-between SEQ ID NO: 4 and SEQ ID NO: 3 is alsofrom the resistant donor. Thus, the introgression fragment comprises theentire chromosome region from SEQ ID NO: 4 to SEQ ID NO: 3.

In another aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 11, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence in-betweenat least SNP_04 and SNP_02, optionally including the donor SNPnucleotide for one or both of these SNP markers. So optionally theintrogression fragment comprises SEQ ID NO: 4 and SEQ ID NO: 2 and thenucleotide sequence in-between SEQ ID NO: 4 and SEQ ID NO: 2 is alsofrom the resistant donor. Thus, in this aspect, the entire chromosomeregion from SEQ ID NO: 2 to SEQ ID NO: 4 is from the donor and comprisesSEQ ID NO: 2, 3 and 4.

In another aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 11, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence in-betweenat least SNP_01 and SNP_03, optionally including the donor SNPnucleotide for one or both of these SNP markers. So optionally theintrogression fragment comprises SEQ ID NO: 1 and SEQ ID NO: 3 and thenucleotide sequence in-between SEQ ID NO: 1 and SEQ ID NO: 3 is alsofrom the resistant donor. Thus, in this aspect, the entire chromosomeregion from SEQ ID NO: 1 to SEQ ID NO: 3 is from the donor and comprisesSEQ ID NO: 1, 2 and 3.

In another aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 11, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence in-betweenat least SNP_01 and SNP_04, optionally including the donor SNPnucleotide for one or both of these SNP markers. So optionally theintrogression fragment comprises SEQ ID NO: 1 and SEQ ID NO: 4 and thenucleotide sequence in-between SEQ ID NO: 1 and SEQ ID NO: 4 is alsofrom the resistant donor. Thus, in this aspect, the entire chromosomeregion from SEQ ID NO: 1 to SEQ ID NO: 4 is from the donor and comprisesSEQ ID NO: 1, 2, 3 and 4. The introgression fragment present in thedeposited seeds comprises SEQ ID NO: 1, 2, 3 and 4. However, as SNP_03is most closely linked to the resistance phenotype, the introgressionfragment can be reduced in size, while still retaining the QTL. Thus,for example, SEQ ID NO: 1, or SEQ ID NO: 1 and 2, may be removed throughrecombination within the introgression fragment and replaced bycultivated melon sequences. Or SEQ ID NO: 4 may be removed throughrecombination within the introgression fragment and replaced bycultivated melon sequences. Or e.g. SEQ ID NO: 1 and SEQ ID NO: 4 may beremoved through recombination on both sides of the introgressionfragment. Likewise e.g. SEQ ID NO: 1 and SEQ ID NO: 2 on one side andSEQ ID NO: 4 on the other side of the fragment may be removed throughrecombination.

In another aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 12, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises a Guanine (G) at nucleotide 101 of SEQID NO: 6 or at the equivalent nucleotide of a sequence comprisingsubstantial sequence identity to SEQ ID NO: 6. In a further aspect theintrogression fragment further comprises a Cytosine (C) at nucleotide101 of SEQ ID NO: 5 or at the equivalent nucleotide of a sequencecomprising substantial sequence identity to SEQ ID NO: 5 and/or theintrogression fragment further comprises a Thymine (T) at nucleotide 101of SEQ ID NO: 7 or at the equivalent nucleotide of a sequence comprisingsubstantial sequence identity to SEQ ID NO: 7.

In one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 12, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence between atleast SNP_06 and SNP_05, optionally including the donor SNP nucleotidefor one or both of these SNP markers. So optionally the introgressionfragment comprises SEQ ID NO: 6 and SEQ ID NO: 5 and the nucleotidesequence in between SEQ ID NO: 6 and SEQ ID NO: 5 is also from theresistant donor. Thus, the introgression fragment comprises the entirechromosome region from SEQ ID NO: 6 to SEQ ID NO: 5.

In one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 12, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence between atleast SNP_06 and SNP_07, optionally including the donor SNP nucleotidefor one or both of these SNP markers. So optionally the introgressionfragment comprises SEQ ID NO: 6 and SEQ ID NO: 7 and the nucleotidesequence in between SEQ ID NO: 6 and SEQ ID NO: 7 is also from theresistant donor. Thus, the introgression fragment comprises the entirechromosome region from SEQ ID NO: 6 to SEQ ID NO: 7.

In yet one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a donor on chromosome 12, whichintrogression fragment confers the ToLCNDV resistance, whereby theintrogression fragment comprises the donor genomic sequence between atleast SNP_05 and SNP_07, optionally including the donor SNP nucleotidefor one or both of these SNP markers. So optionally the introgressionfragment comprises SEQ ID NO: 5 and SEQ ID NO: 7 and the nucleotidesequence in between SEQ ID NO: 5 and SEQ ID NO: 7 is also from theresistant donor. Thus, the introgression fragment comprises the entirechromosome region from SEQ ID NO: 5 to SEQ ID NO: 7. The introgressionfragment present in the deposited seeds comprises SEQ ID NO: 5, 6 and 7.However, as SNP_06 is most closely linked to the resistance phenotype,the introgression fragment can be reduced in size, while still retainingthe QTL. Thus, for example SEQ ID NO: 5 and/or SEQ ID NO: 7 may beremoved through recombination within the introgression fragment andreplaced by cultivated melon sequences.

As mentioned before, plants, plant parts and plant cells comprising botha introgression fragment on chromosome 11 and an introgression fragmenton chromosome 12 are encompassed herein.

Plants comprising plant cells according to the invention are anotherembodiment of the invention.

The melon plant according to the invention may be an inbred line, anopen pollinated variety (OP) or an F1 hybrid variety.

In one aspect the F1 hybrid comprises the introgression fragment onchromosome 11 and/or 12 in heterozygous form, i.e. produced by crossingtwo inbred parent lines, one of which possesses the introgressionfragment on chromosome 11 and/or 12 (preferably in homozygous form,although not necessarily) and collecting the F1 hybrid seeds from saidcross. The F1 hybrid may also comprise the introgression fragment onchromosome 11 and/or 12 in homozygous form, i.e. produced by crossingtwo inbred parent lines, each comprising the introgression fragment onchromosome 11 and/or 12 in homozygous or heterozygous form.

In a preferred aspect, the melon plant is an F1 hybrid and comprises theintrogression fragment on chromosome 11 in homozygous form. Optionally,the F1 hybrid further comprises the introgression fragment on chromosome12 in heterozygous or, preferably, in homozygous form. Such an F1 hybridis preferably produced by crossing two inbred lines, each of whichcomprises the introgression fragment in chromosome 11 and theintrogression fragment on chromosome 12, preferably both in homozygousform. Such inbred lines are also encompassed herein.

The melon plant according to the invention may be of any type.Preferably it has good agronomic and good fruit quality characteristics,such as e.g. large average fruit size (at least 500 g, 600 g, 700 g, 800g, 900 g, 1000 g or more), high average brix of the fruits (e.g. anaverage refractometer % total soluble solids of at least 10%, 12%, 14%,16%, 18% or more), many fruits being produced per plant, firm fruitflesh, etc.

The cultivated melon plants and plant cells belong in one aspect to oneof the following species: C. melo var. cantalupensis, C. melo var.inodorous or C. melo var. reticulatus.

In one aspect the cultivated melon of the invention comprising one ortwo introgression fragments conferring ToLCNDV resistance, and thecultivated melon comprises one or more or all of the followingcharacteristics: an average TSS at maturity of at least 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%; an attachment of the peduncle at fruitmaturity which is not ‘very weak’ (UPOV characteristic 39 in TG/104/5Rev., UPOV Code: CUCUM_MEL, found on the website UPOV.int); a shelf lifeof the fruit which is not ‘very short’ (UPOV characteristic 68 inTG/104/5 Rev., UPOV Code: CUCUM_MEL, found on the website UPOV.int); afruit shape in longitudinal section which is not elongated (UPOVcharacteristic 28 in TG/104/5 Rev. UPOV Code: CUCUM_MEL, found on thewebsite UPOV.int).

The average % TSS (or brix) can be measured as known in the art, e.g.using a digital hand refractometer and measuring the TSS of severalmature fruits of a line or variety. See for example Burger et al. 2003,J American Soc Hort Science 128(4): 537-540 on page 538 at Measurementof TSS.

Also other resistances may be introduced into the melon plants of theinvention, such as resistance to one or more of the following diseases:Bacterial Wilt, Root Rot, Crown Blight, Melon Rust, Powdery Mildew,Verticillum Wilt, Sulphur Bum, Scab, Watermelon Mosaic, Downy Mildew,Fusarium oxysporum fsp. melonis (Fom) race 0, Fusarium oxysporum fsp.melonis (Fom) race 1, Fusarium oxysporum fsp. melonis (Fom) race 2,Fusarium oxysporum fsp. melonis (Fom) race 1.2, Fusarium Wilt

R2, Root Knot (Nematode), Anthracnose, Cucumber Mosaic, and SquashMosaic, and/or resistance to one or more of the following pests: Aphidresistance, Pickle Worm, Darkling Ground Beetle, Banded Cucumber Beetle,Mite, Western Spotted Cucumber Beetle, Melon Leafhopper, Melon Worm,Western Striped Cucumber Beetle or Melon Leafminer. Other resistancegenes, against pathogenic viruses, fungi, bacteria or pests may also beintroduced.

A specific aspect of the invention concerns plants or plant cellscomprising an introgression fragment according to the invention whichintrogression fragment is obtainable from (derivable from, or as presentin) seeds deposited under Accession number NCIMB 42625 or from progenythereof The seeds deposited are cultivated melon plants comprising bothintrogression fragments in homozygous form, with the donor nucleotidebeing present in homozygous form for SNP_01, SNP_02, SNP_03, SNP_04,SNP_05, SNP_06 and SNP_07. For the ToLCNDV resistance conferring QTL11marker SNP_03 was found to be most significantly and for QTL12 SNP_06was found to be most significant. This means that the size of the donorintrogression can be reduced both for QTL11 and for QTL12, by selectingrecombinants having smaller introgression fragment sizes for QTL11and/or QTL12, i.e. sub-fragments of the fragments present in thedeposited seeds. For example, plants comprising sub-fragments of theseintrogression fragments (wherein said sub-fragments still comprise theToLCNDV resistance conferring QTL11 or 12), comprising the donor SNPgenotype for SNP_03 (regarding QTL11) or comprising the donor SNPgenotype SNP_06 (regarding QTL12), but having the SNP genotype of therecurrent parent for one or more or all of the other SNPs can begenerated in ways known to the skilled person. Thus, melon plantscomprising sub-fragments comprising only one of the donor SNPs (i.e. oneof SNP_01, SNP_02, SNP_03 and SNP_04 for QTL11 and one of SNP_05, SNP_06and SNP_07 for QTL12) but still retaining the resistance conferring QTLcan be generated by the skilled person. It is even possible for theskilled person to remove all donor SNPs and still retain the QTL. Onecan sequence the chromosome 11 and/or 12 region to identify or determineif a plant has an introgression fragment or sub-fragment as present in(or obtainable from) seeds deposited under Accession number NCIMB 42625.Also, whether the QTL11 and/or 12 is still present on a sub-fragmentwhich does not contain all of the donor SNPs or which even does notcontain any of the donor SNPs anymore can be easily tested by carryingout a ToLCNDV resistance assay as described herein.

An introgression fragment can be identified by various methods, such asthe polymorphic markers provided herein (SNP markers, polymorphicbetween the donor and the recurrent parent), chromosome painting orsequencing the melon genome and identifying the chromosome parts onchromosome 11 and/or 12 which are introgressions from a specific donor.The nucleotide sequence of the introgression fragment will be specificand unique for a donor accession that has been used to introgress thetrait. For example to identify the introgression fragment from thespecific donor of the invention e.g., whole genome sequencing of thegenome of the seeds deposited can be done. This has, for example, alsobeen done in tomato, where genome sequences of tomato inbreeding linesand hybrids identified introgression fragments on chromosome 6 andchromosome 9 of specific wild accessions. See Lin et al., NatureGenetics published Oct. 12, 2014, doi:10.1038/ng.3117, page 5, FIG. 4.

Apart from melon plants, also seeds from which such plants can be grownare provided herein.

A further aspect of the present invention, therefore, concerns melonseeds comprising an introgression fragment from chromosome 11 and/or 12of a ToLCNDV resistant donor plant, wherein the introgression fragmentcomprises the sequence of the ToLCNDV resistant donor melon plantin-between SNP_01 and SNP_04, or in-between SNP_01 and SNP_03, orin-between SNP O1 and SNP_02, or in-between SNP_02 and SNP_04, orin-between SNP_02 and SNP_03, or in-between SNP_03 and SNP_04 forchromosome 11; and/or wherein the introgression fragment comprises thesequence of the ToLCNDV resistant donor melon plant in-between SNP_05and SNP_07, or in-between SNP_05 and SNP_06, or in-between SNP_06 andSNP_07 for chromosome 12. It is understood that the introgressionfragment comprises the resistance conferring QTL. Thus, if theintrogression fragment is transferred into a ToLCNDV susceptible melonline or variety, especially in homozygous form, then the otherwiseToLCNDV susceptible line or variety will become resistant or highlyresistant against ToLCNDV as described. In one aspect the cultivatedmelon plant comprising an introgression fragment comprising QTL11 and/orQTL12 has an average disease score of 9.0 (no symptoms), while thesusceptible control shows the expected symptoms.

In another aspect ToLCNDV virus is not detected in the upper parts ofthe inoculated ToLCNDV resistant plants or is only detected at anextremely low level, see examples. Thus plants comprising QTL11 and/orQTL12 (e.g. a donor plant or a plant comprising one or bothintrogression fragments) comprise a significantly lower average ToLCNDVvirus level (or virus titer) in the upper leaves at e.g. 15 dpi (dayspost infection), 20 dpi, 25 dpi, 30 dpi and/or 35 dpi (or later) thanthe susceptible control line or variety. “Significantly lower” refersherein to an average virus level, detectable by e.g. qPCR, which is atleast 1000 fold lower, more preferably at least 5000 fold, or at least10000 fold, or at least 50000 fold, or at least 100000 fold, or at least1000000 fold, lower than in the susceptible control tissue.Quantification of ToLCNDV levels is for example described in Simon etal. (Plant Disease January 2018, Volume 102, Number 1, Pages 165-171).In one aspect the average Ct (Cycle threshold) value of the resistantplant line is at least 2 times the value of the susceptible line orvariety, or at least 2.5 times or 3.0 times the value. In one aspect theaverage Ct value of the resistant plant line is at least 30, 31, 32, 33,34, 35, 36, 37 or 38, while the average Ct value of the susceptible lineor variety is 15 or less, 14 or less, e.g. 13 or less, 12 or less, 11 orless, 10 or less. See e.g. world wide web atbitesizebio.com/24581/what-is-a-ct-value/or references cited therein.Ct-values are inverse to the amount of target nucleic acid (in this caseToLCNDV virus DNA) that is in a sample, and correlate to the number ofcopies in a sample. Lower Ct-values indicate high amounts of targetednucleic acid, while higher Ct-values mean lower amounts of targetnucleic acid.

In one embodiment of the invention the seeds comprise an introgressionfragment from chromosome 11 of a ToLCNDV resistant donor plant, whereinthe introgression fragment comprises the sequence of the ToLCNDVresistant donor melon plant in-between SNP_02 and SNP_04, or in-betweenSNP_02 and SNP_03, or in-between SNP_03 and SNP_04.

Another embodiment of the invention concerns melon seeds obtainable orobtained from plants according to the invention, or seeds comprisingplant cells according to the invention.

A further aspect of the present invention concerns melon plant fruitscomprising an introgression fragment from chromosome 11 and/or 12 of aToLCNDV resistant donor plant, wherein the introgression fragmentcomprises the sequence of the ToLCNDV resistant donor melon plantin-between SNP_01 and SNP_04, or in-between SNP_01 and SNP_03, orin-between SNP O1 and SNP_02, or in-between SNP_02 and SNP_04, orin-between SNP_02 and SNP_03, or in-between SNP_03 and SNP_04 forchromosome 11; and/or wherein the introgression fragment comprises thesequence of the ToLCNDV resistant donor melon plant in-between SNP_05and SNP_07, or in-between SNP_05 and SNP_06, or in-between SNP_06 andSNP_07 for chromosome 12.

Another embodiment of the invention concerns melon fruits obtainable orobtained from plants according to the invention, or fruits comprisingplant cells according to the invention.

The preferred and further embodiments described herein for melon plantcells or melon plants according to the invention are applicable to alsorepresent preferred and further embodiments of the melon fruits of melonplants according to the invention, accordingly.

A further aspect of the present invention concerns melon plantpropagation material comprising an introgression fragment fromchromosome 11 and/or 12 of a ToLCNDV resistant donor plant, wherein theintrogression fragment comprises the sequence of the ToLCNDV resistantdonor melon plant in-between SNP_01 and SNP_04, or in-between SNP_01 andSNP_03, or in-between SNP_01 and SNP_02, or in-between SNP_02 andSNP_04, or in-between SNP_02 and SNP_03, or in-between SNP_03 and SNP_04for chromosome 11; and/or wherein the introgression fragment comprisesthe sequence of the ToLCNDV resistant donor melon plant in-betweenSNP_05 and SNP_07, or in-between SNP_05 and SNP_06, or in-between SNP_06and SNP_07 for chromosome 12.

Another embodiment of the invention concerns melon plant propagationmaterial obtainable or obtained from plants according to the invention,or melon plant propagation material comprising plant cells according tothe invention.

The preferred and further embodiments described herein for plant cellsor plants according to the invention are applicable to also representpreferred and further embodiments of the propagation material of melonplants according to the invention, accordingly.

The term “propagation material” comprises those components of the plantwhich are suitable for generating progeny via the vegetative (agamic) orgenerative (gamic, sexual) route. Suitable for vegetative propagationare, for example, cuttings, in vitro tissue, cell, protoplast, embryo orcallus cultures, micropropagation methods, or roots. Other propagationmaterial includes, for example, fruits, seeds, seedling, comprising thedescribed one or two introgression fragments conferring ToLCNDVresistance etc. The propagation material in one aspect takes the form ofcuttings which are propagated by grafting to another rootstock or invitro tissue culture material, in particular embryo cultures. Inparticular preferred is propagation material in the form of in vitrotissue culture material, particularly in vitro embryo cultures.

A further embodiment of the invention concerns a method for producing aToLCNDV resistant melon plant comprising the following steps

-   -   a) Selecting a ToLCNDV resistant donor plant    -   b) Crossing the donor plant selected in step a) with a recurrent        plant sensitive to ToLCNDV    -   c) Obtaining seeds from the plants crossed in step b) and        optionally    -   d) Selfing the plant grown from the seeds one or more time or        backcrossing the plant grown from the seed to a recurrent plant        sensitive to TolCNDV one or more times to obtain selfing or        backcross progeny, and optionally    -   e) Testing if (or testing which of) the plants grown from the        seeds obtained in step c) or the selfing or backcross plants of        step d) are resistant to ToLCNDV and/or if the plants comprise        one or more of the SNPs from the donor plant selected from the        group of SNP_01, SNP_02, SNP_03 and SNP_04; and/or SNP_05,        SNP_06 and SNP07.

The method optionally further comprises the step of selecting a ToLCNDVresistant plant comprising QTL11 and/or QTL12, preferably in homozygousform. Optionally the plant comprising one or more of the SNP markers isselfed to generate a homozygous plant.

A ToLCNDV resistant donor plant in step a) in the method for producing aToLCNDV resistant melon plant according to the invention can be selectedby infection of melon plants with ToLCNDV and determining the level ofsymptoms of ToLCNDV infected melon plants as described elsewhere herein.In one aspect the donor plant has an average disease score of 9.0.Optionally, the donor plant comprises one or more of the resistant SNPgenotype for one or more or all of SNPs selected from SNP_01, SNP02,SNP_03 and SNP_04; and/or for one or more or all of SNPs selected fromSNP_05, SNP_06 and SNP_07. In one aspect the donor has an averagedisease score of 9.0 and comprises the resistant genotype of Table 3 forat least SNP_03 and/or SNP_06. In another aspect the donor has anaverage ToLCNDV disease score of 9.0 and comprises the resistantgenotype for SNP_02, SNP_03 and SNP_04, optionally also for SNP_01. In afurther aspect the donor has an average ToLCNDV disease score of 9.0 andcomprises the resistant SNP genotype for SNP_01, SNP_02, SNP_03, SNP_04,SNP_05 and SNP_06, and optionally SNP_07. Preferably the donor ishomozygous for the SNP genotypes. Optionally the donor may be selfed toselect a donor plant which is homozygous. In a further aspect the donorplant comprises the same sequence in-between SNP_01 and SNP_04 and/orin-between SNP_05 and SNP_06 (optionally in-between SNP_05 and SNP_07)as the fragment introgressed in seeds deposited under accession numberNCIMB 42625. The donor may be a wild melon or wild relative of melon.The donor may be the same donor as used to introgress QTL11 and QTL12 inseeds of NCIMB 42625. In one aspect ToLCNDV virus particles are very low(e.g. average Ct-value of at least 30 or more) or not detectable in theupper leaves of the donor plant following infection, e.g. using e.g.qPCR or dot blot as described e.g. in the Examples.

Another embodiment of the invention concerns a method for producing aToLCNDV resistant melon plant comprising the following steps:

-   -   a) Selecting a ToLCNDV resistant cultivated melon plant        comprising an introgression fragment on chromosome 11 and/or 12,        said introgression confers ToLCNDV resistance as described        herein,    -   b) Crossing the melon plant selected in step a) with another        melon plant, e.g. a melon plant sensitive to ToLCNDV or a plant        not resistant to ToLCNDV, e.g. a plant comprising an average        TolCNDV disease score of 4.0 or less, preferably less than 4.0        or less than 3.0,    -   c) Obtaining seeds from the plants crossed in step b) and        optionally    -   d) Selfing the plant grown from the seeds one or more time or        backcrossing the plant grown from the seed to a plant sensitive        to ToLCNDV one or more times to obtain selfing or backcross        progeny, and optionally    -   e) Testing if (or which of) the plants grown from the seeds        obtained in step c) or the selfing or backcross plants of        step d) are resistant to ToLCNDV and/or if the plants comprise        one or more of the SNPs from the donor plant selected from the        group of SNP_01, SNP_02, SNP_03 and SNP_04; and/or SNP_05,        SNP_06 and SNP_07.

The method optionally further comprises the step of selecting a ToLCNDVresistant plant comprising QTL11 and/or QTL12, preferably in homozygousform.

A ToLCNDV resistant cultivated plant in step a) in the method forproducing a ToLCNDV resistant melon plant according to the invention canbe selected by infection of melon plants with ToLCNDV and determiningthe level of symptoms of ToLCNDV infected melon plants as describedelsewhere herein. In one aspect the cultivated plant has an averagedisease score of at least 5.0, 6.0, 7.0, 8.0 or 9.0. Optionally, thecultivated plant comprises one or more of the resistant donor SNPgenotypes for one or more or all of SNPs selected from SNP_01, SNP_02,SNP_03 and SNP_04; and/or for one or more or all of SNPs selected fromSNP_05, SNP_06 and optionally SNP_07. In one aspect the cultivated plantis a cultivated melon plant comprising an introgression fragment onchromosome 11 and/or 12 as described throughout the specification. Inone aspect the plant in step a) may be a plant grown from seedsdeposited under accession number NCIMB 42625 or a progeny plant thereofwhich progeny plant retains QTL11 and/or QTL12. It may, thus, be a plantwhich comprises the same introgression fragment on chromosome 11 and/or12 or a smaller introgression fragment on chromosome 11 and/or 12 thanthe one found in the deposited seeds.

In one aspect of the methods for producing a ToLCNDV resistant melonplant according to the invention the ToLCNDV resistant donor plant orcultivated melon plant in step a) comprises a fragment on chromosome 11and/or 12 wherein the fragment comprises the sequence of the ToLCNDVresistant donor melon plant in-between SNP_01 and SNP_04, or in-betweenSNP_01 and SNP_03, or in-between SNP_01 and SNP_02, or in-between SNP_02and SNP_04, or in-between SNP_02 and SNP_03, or in-between SNP_03 andSNP_04 for chromosome 11; and/or wherein the fragment comprises thesequence of the ToLCNDV resistant donor melon plant in-between SNP_05and SNP_07, or in-between SNP_05 and SNP_06, or in-between SNP_06 andSNP_07 for chromosome 12.

In one embodiment of the invention the method for producing a ToLCNDVresistant melon plant according to the invention is used for producing aplant according to the invention. The preferred and further embodimentsas described herein for the plants according to the invention areapplicable accordingly to the method for producing a ToLCNDV resistantmelon plant according to the invention.

Plants obtainable or obtained by a method for producing a ToLCNDVresistant melon plant according to the invention are also an embodimentof the invention.

A further embodiment of the invention concerns methods for producingmelon fruits and optionally melon seeds comprising the following steps

-   -   a) growing a ToLCNDV resistant melon plant comprising two        chromosomes 11 each having an introgression fragment from        chromosome 11 of a ToLCNDV resistant donor plant, the        introgression fragment comprising the sequence of the donor        plant for SNP_03, or in-between SNP_03 and SNP_04, or in-between        SNP_02 and SNP_03; or the introgression fragment comprising the        resistant donor genotype for one or more or all of SNP_01,        SNP_02, SNP_03 and SNP04;    -   b) harvesting the fruits of the melon plants grown in step a),        and optionally    -   c) collecting the seeds from the fruits obtained in step b).

The melon plant of step a) is a plant as described in the specification,comprising QTL11 as described earlier, especially QTL11 is in homozygousform so that the phenotype is expressed. Optionally, the melon plant mayfurther comprise an introgression fragment on chromosome 12, also asdescribed earlier. Thus the plant of step a) may further comprise one ortwo chromosomes 12 having an introgression fragment on chromosome 12 ofa ToLCNDV resistant donor plant, said introgression fragment comprisingthe sequence of the donor plant for SNP_05 and/or SNP_06 and/or SNP_07,or in-between SNP_05 and SNP07, or in-between SNP_05 and SNP_06, orin-between SNP_06 and SNP_07.

In one aspect the cultivated melon plant of step a) is an F1 hybridplant, and melon fruits for commercial use are produced.

In a preferred embodiment of the invention the melon plants of steps a)of the method for producing melon fruits or seeds according to theinvention has the specific characteristics described as differentembodiments of the plants according to the invention. The differentembodiments as described herein for the plants according to theinvention are applicable accordingly to the method for producing ahybrid melon seed according to the invention.

Seeds obtainable the method for producing melon seeds according to theinvention are also an embodiment of the invention.

Another embodiment of the invention concerns methods for producinghybrid melon seeds, especially F1 hybrid seeds, comprising the followingsteps

-   -   a) providing a first ToLCNDV resistant inbred melon plant        comprising two chromosomes 11 each having an introgression        fragment from chromosome 11 of a ToLCNDV resistant donor plant,        the introgression fragment comprising QTL11 and the sequence of        the donor plant for SNP_03, or in-between SNP_03 and SNP_04, or        in-between SNP_02 and SNP_03; or the introgression fragment        comprising QTL11 and the resistant donor genotype for one or        more or all of SNP_01, SNP_02, SNP_03 and SNP_04;    -   b) providing a second ToLCNDV resistant inbred melon plant        comprising two chromosomes 11 each having an introgression        fragment from chromosome 11 of a ToLCNDV resistant donor plant,        the introgression fragment comprising QTL11 and the sequence of        the donor plant for SNP03, or in-between SNP_03 and SNP_04, or        in-between SNP_02 and SNP_03; or the introgression fragment        comprising QTL11 and the resistant donor genotype for one or        more or all of SNP_01, SNP_02, SNP_03 and SNP_04;    -   c) crossing the inbred melon plant provided in step a) with the        inbred melon plant provided in step b)    -   d) collecting seeds obtained from the cross of step c).

In one aspect the inbred plant of step a) or of step b) is male sterile,so that only cross pollination occurs in step c), generating F1 hybridseeds in step d).

Optionally the inbred plant in step a) or in step b) comprises inaddition two chromosomes 12, each of which comprises an introgressionfragment having QTL12, as described. Thus in one aspect the plant instep a) or in step b) comprises QTL11 in homozygous form and furthercomprises QTL12 homozygous form. The F1 hybrid seeds therefore compriseQTL11 in homozygous form and QTL12 in heterozygous form.

In another aspect both the plant in step a) and the plant in step b)comprise QTL11 in homozygous form and QTL12 in homozygous form. The F1hybrid seeds of step c) therefore comprise both QTLs in homozygous form.

The hybrid seed, especially F1 hybrid seed, collected are also anembodiment of the invention. These are the seeds that are sold forcommercial melon fruit production. Plants grown from these seeds will beresistant or highly resistant against ToLCNDV in the fruit productionfields.

In a different embodiment of the invention concerns methods forproducing hybrid melon seeds, especially F1 hybrid seeds, comprising thefollowing steps

-   -   a) providing a first ToLCNDV resistant inbred melon plant        comprising two chromosomes 12 each having an introgression        fragment from chromosome 12 of a ToLCNDV resistant donor plant,        the introgression fragment comprising QTL12 and the sequence of        the donor plant for SNP_06, or in-between SNP_05 and SNP07, or        in-between SNP_05 and SNP06, or in-between SNP_06 and SNP_07; or        the introgression fragment comprising QTL12 and the resistant        donor genotype for one or more or all of SNP_05 and SNP_06 and        SNP_07;    -   b) providing a second inbred melon plant;    -   c) crossing the inbred melon plant provided in step a) with the        inbred melon plant provided in step b)    -   d) collecting seeds obtained from the cross of step c).

Optionally the second inbred melon plant in step b) is also a ToLCNDVresistant inbred melon plant comprising two chromosomes 12 having anintrogression fragment from chromosome 12 of a ToLCNDV resistant donorplant, the introgression fragment comprising QTL12 and the sequence ofthe donor plant for SNP_06, or in-between SNP_05 and SNP_07 orin-between SNP_05 and SNP_06, or in-between SNP_06 and SNP_07; or theintrogression fragment comprising QTL12 and the resistant donor genotypefor one or more or all of SNP_05 and SNP_06 and SNP_07.

In one aspect the inbred plant of step a) or of step b) is male sterile,so that only cross pollination occurs in step c), generating F1 hybridseeds in step d).

The F1 hybrid seeds collected in step d) comprise QTL12 in eitherheterozygous form or in homozygous form. As QTL12 is partially dominant,the ToLCNDV resistance score will be slightly higher in the homozygousplant than in the plant heterozygous for QTL12.

The hybrid seed, especially F1 hybrid seed, collected are also anembodiment of the invention. These are the seeds that are sold forcommercial melon fruit production. Plants grown from these seeds will beresistant against ToLCNDV in the fruit production fields.

“Inbred plant” or “inbred line” shall mean in connection with thepresent invention plants which have undergone several generations ofselfing and are highly uniform in respect to their genetic setup andphenotypic appearance.

In a preferred embodiment of the invention the inbred lines of steps a)and b) of the method for producing hybrid melon seeds according to theinvention has the specific characteristics described as preferred andfurther embodiments of the plants according to the invention. Thepreferred and further embodiments as described herein for the plantsaccording to the invention are applicable accordingly to the method forproducing a hybrid melon seed according to the invention.

Hybrid seeds obtainable or obtained by the method for producing hybridmelon seeds according to the invention are also an embodiment of theinvention.

A further embodiment of the present invention are methods for producinga melon fruit comprising the following step

-   -   a) growing a F1 hybrid plant described above, i.e. comprising        QTL11 in homozygous form and/or QTL12 in heterozygous or        homozygous form in its genome, and optionally    -   b) harvesting the fruits produced by the plants grown in step        a).

The term “fruit” in its botanical meaning is commonly understood to be aseed bearing structure developed from the ovary of angiosperm flowers.

Melon fruits obtainable or obtained by a method for producing a melonfruit according to the invention are also an embodiment of theinvention.

Melon donor plants being resistant to ToLCNDV due to the presence ofQTL11 and/or QTL12 in their genome can be identified with the aid of theSNP markers, in particular one or more or all of SNP_01, SNP_02 SNP_03,SNP_04 and/or one or more of SNP_05, SNP_06 and SNP_07 disclosed herein.The present invention therefore for the first time enables a personskilled in the art to identify donor plants from which an introgressionfragment conferring ToLCNDV resistance to melon plants can betransferred into recurrent melon plants.

Likewise, the invention provides for the first time cultivated melonplants, plant parts and cells, comprising in their genome anintrogression fragment on chromosome 11 and/or 12, which confers ToLCNDVresistance to an otherwise susceptible melon plant.

A further embodiment of the invention therefore pertains the use of oneor more or all of SNP_01, SNP_02, SNP_03, SNP_04 and/or one or more orall of SNP_05, SNP_06 and SNP_07 for identification of a ToLCNDVresistant melon plant or parts thereof (such as cells, fruits, leaves).

Preferably the use pertains to the identification of a ToLCNDV resistantdonor melon plant or plant part and/or a cultivated melon plant or plantpart.

Another embodiment is the use of one or more or all of SNP_01, SNP_02,SNP_03, SNP_04 and/or of one or more or all of SNP_05, SNP_06 and SNP_07for introgression of ToLCNDV resistance into a ToLCNDV susceptible melonplant, especially a cultivated melon line or variety.

Also an embodiment of the invention is the use of one or more or all ofSNP_01, SNP_02, SNP_03, SNP_04 and/or one or more or all of SNP_05,SNP_06 and SNP_07 in breeding ToLCNDV resistant melon plants.

Also provided is a method of screening (or selecting) plants or plantparts (such as plant tissues, cells, etc.) or DNA derived therefrom forthe presence of a fragment on chromosome 11 and/or 12 conferring ToLCNDVresistance. The method comprises the steps of:

-   -   i) screening the genomic DNA for the SNP genotype of one or more        or all of SNP_01, SNP_02, SNP_03, SNP_04 and/or one or more or        all of SNP_05, SNP_06 and SNP07;    -   ii) and optionally selecting a plant or plant part which        comprise the resistant donor genotype of one or more or all of        SNP_01, SNP_02, SNP_03, SNP_04 and/or for one or more or all of        SNP05, SNP_06 and SNP_07.

Such screening can be carried out using various methods, such as SNPgenotyping assays (e.g. KASP assays, TaqMan assays, a High ResolutionMelting (HRM) assays, SNP-genotyping arrays such as e.g. Fluidigm,Illumina, etc., or DNA sequencing may equally be used.

As clear throughout the description, screening of plants, plant parts,or genomic DNA thereof, can be used for various purposes, for exampleidentification of ToLCNDV resistant donors, or as part of a breedingprogram, e.g. identification and/or selection of plants or plant partscomprising QTL11 and/or QTL12, or for checking whether competitors aremaking use of the instant invention and produce inbred lines or hybridscomprising QTL11 and/or QTL12.

Also provided is a method for growing the plants of the invention, e.g.in areas where ToLCNDV virus infestation occurs, comprising sowing theseeds or planting seedlings into a growing area (e.g. a field orprotected environment such as a glasshouse or tunnel) and optionallyharvesting the fruits.

Cells or tissues comprising an introgression fragment on chromosome 11and/or 12 as described herein may consist of or comprise non-propagatingcells or tissues, or non-regenerable cells or tissues, or alternativelypropagating or regenerable cells or tissues. They may comprise orconsist of photosynthetic or non-photosynthetic cells. Vegetative plantparts, cells and tissues, such as harvested fruits or fruit flesh partsare non-photosynthetic parts and tissues. Cells and tissues may be incell or tissue cultures.

Harvested fruits or processed fruits are also encompassed herein, e.g.slices, blocks or cubes of melon fruit flesh, e.g. in a container.

In one aspect the plants, plant parts and plant cells according to theinvention are not exclusively obtained by means of an essentiallybiological process as defined by Rule 28 (2) EPC (European PatentConvention).

In one aspect the plants are non-GMO (not genetically modified).

The instant QTL11 is a different QTL than the one described inWO2017/114848, as the location on chromosome 11 is a different location.This is evident when BLASTing the sequences of SNP1 to SNP16 markersagainst the CM3.6.1 genome of melon. The most closely linked markers areMelon_sbg_33761_74 (SEQ ID NO: 7), Melon_sbg_2720_78 (SEQ ID NO: 8) andMelon_sbg_14207_58 (SEQ ID NO: 9), which are located at 33.18 Mb, 33.34Mb and 33.35 Mb of the chromosome. In contrast, the most closely linkedmarker of the instant QTL11, SNP_03, is located at 32.49 Mb. Thislocation is further upwards on chromosome 11, in the region of theirmarker Melon_sbg_55680_17 (SEQ ID NO: 5), located at 32.3 Mb, andMelon_sbg_60684_74 (SEQ ID O: 6) located at 32.6 Mb. However, in RIL-30the introgression fragment is not present in the region corresponding toMelon_sbg_617_42 (SEQ ID NO: 1) to Melon_sbg_33761_74 (SEQ ID NO: 7),while the line is still resistant to ToLCNDV and still contains theirQTL11. The nucleotide sequence of their introgressions will also beentirely different than the nucleotide sequence of the instantinvention, as the donor is a Cucumis melo subsp. agrestis var acidulousaccession, while the instant donor is a Cucumis melo subsp. meloaccession. Cucumis melo subsp. agrestis is phylogenetically in aseparate cluster from Cucumis melo subsp. melo, see for exampleSupplementary Figure S3 of Gur et al. “Genome-WideLinkage-Disequilibrium Mapping to the Candidate Gene Level in Melon(Cucumis melo)”, Scientific Reports, volume 7, Article number: 9770(2017), doi:10.1038/s41598-017-09987-4.

Seed Deposit Information

A representative sample of seeds of a cultivated melon, designatedCucumis melo TOLCHR11-12, comprising two introgression fragments (inhomozygous form), one comprising ToLCNDV resistance introgressed onchromosome 11 and one comprising ToLCNDV resistance introgressed onchromosome 12, was deposited by Nunhems B. V. on 10 Aug. 2016 at theNCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn Aberdeen,Scotland AB21 9YA, UK) according to the Budapest Treaty, under theExpert Solution (EPC 2000, Rule 32(1)). Seeds were given the followingdeposit numbers NCIMB 42625.

The Applicant requests that samples of the biological material and anymaterial derived therefrom be only released to a designated Expert inaccordance with Rule 32(1) EPC or related legislation of countries ortreaties having similar rules and regulation, until the mention of thegrant of the patent, or for 20 years from the date of filing if theapplication is refused, withdrawn or deemed to be withdrawn.

Access to the deposit will be available during the pendency of thisapplication to persons determined by the Director of the U.S. PatentOffice to be entitled thereto upon request. Subject to 37 C.F.R. §1.808(b), all restrictions imposed by the depositor on the availabilityto the public of the deposited material will be irrevocably removed uponthe granting of the patent. The deposit will be maintained for a periodof 30 years, or 5 years after the most recent request, or for theenforceable life of the patent whichever is longer, and will be replacedif it ever becomes nonviable during that period. Applicant does notwaive any rights granted under this patent on this application or underthe Plant Variety Protection Act (7 USC 2321 et seq.).

Description of Sequences

Characters other than G (guanine), A (adenine), T (thymine) and C(cytosine) have the following meaning in the SEQ ID NOs shown in thesequence listing:

R: G or A

Y: T or C

M: A or C

K: G or T

S: G or C

W: A or T

H: A or C or T

B: G or T or C

V: G or C or A

D: G or A or T

N: G or A or T or C

-   SEQ ID NO 1: Sequence of the ToLCNDV resistant donor plant    comprising SNP_01.-   SEQ ID NO 2: Sequence of the ToLCNDV resistant donor plant    comprising SNP_02.-   SEQ ID NO 3: Sequence of the ToLCNDV resistant donor plant    comprising SNP_03.-   SEQ ID NO 4: Sequence of the ToLCNDV resistant donor plant    comprising SNP_04.-   SEQ ID NO 5: Sequence of the ToLCNDV resistant donor plant    comprising SNP_05.-   SEQ ID NO 6: Sequence of the ToLCNDV resistant donor plant    comprising SNP_06.-   SEQ ID NO 7: Sequence of the ToLCNDV resistant donor plant    comprising SNP_07.-   SEQ ID NO 8-10: SNP_01 FAM Allele Primer, SNP_01 VIC allele primer,    SNP_01 Common Primer-   SEQ ID NO 11-13: SNP_02 FAM Allele Primer, SNP_02 VIC allele primer,    SNP_02 Common Primer-   SEQ ID NO 14-16: SNP_03 FAM Allele Primer, SNP_03 VIC allele primer,    SNP_03 Common Primer-   SEQ ID NO 17-19: SNP_04 FAM Allele Primer, SNP_04 VIC allele primer,    SNP_04 Common Primer-   SEQ ID NO 20-22: SNP_05 FAM Allele Primer, SNP_05 VIC allele primer,    SNP_05 Common Primer-   SEQ ID NO 23-25: SNP_06 FAM Allele Primer, SNP_06 VIC allele primer,    SNP_06 Common Primer-   SEQ ID NO 26-28: SNP_07 FAM Allele Primer, SNP_07 VIC allele primer,    SNP_07 Common Primer

SEQ ID NO: 1 gctaatgaac tcgggttttg aaacgctcag aggattatcgttaccggaaa catgaacgga agtccatgtt ttgacaaacgaactaatgga cgacgcgtcg aaaatcttgt gcgacatgagtaagccaaca gtaatgcctc cacattgaaa cgtcgtggtttggatgagta caatacatcc tttgttagcg agatgaaaag g SEQ ID NO: 2gtttgttttg atcaaaaaaa tcaaacattt aagtaaataatatactacat taaatgtgta agtgagtcta gcagtatacattccgtcgat gtactaaact gaattcaaag agtttatgcatataagtgaa tgaatatccc aaaacttatc aatttcagaagaagagaaaa aactaggttt ccttatcctt tttgtgagcc a SEQ ID NO: 3ctcgtagagc atttgaacgc cacagtggaa ctggccgtgggtaagtgtct gctgtctgtt cttttaaatt ttgttgtgcacactcctgct gcatattcta tgccattttg acacttcattcctgatttta tgcctagaaa tgaatttaaa cgagaagggtctggtcgtgg aaattgggga aggtcaactg acgaatttgc t SEQ ID NO: 4tctctctggt ggataactca tgtaatggtc ttgtctttatttgtaagtgt gcttttttta ctcgttgtga tggcatattcaatcctatga ctaatgagtt tttccaaatt cctcgaggtgaattagatgg tgatatttac tcctacgaat taggttttagccctacaaca aagcaataca aattgtttcg agttaccgaa t SEQ ID NO: 5atacaaccca tcaaccaaaa tcttccaact aattatagagactttgataa aaggaaaaaa aagagtaaca aatagcgatgcgaggaaagg gacgatggcc cgtacatatt agcatgctcataaacaaatt caaaaagccc aacaaagacg aagaaaaataacgaaaacca tgatggcaca aagcattgtc aagtctcgga a SEQ ID NO: 6tgacaacagc tttcaccacc aatcaataaa atgaggatggattcaagatt tatgccaaca ccctgatccc aaattgaagatgccaaacaa agggacacag gaatcattgt ctaagaaattgtaaaaaccc cacaatgaaa acttctatca aagacacgggaatctacagt ttgtacagtc acttgcacaa gcatcttcaa a SEQ ID NO: 7atcgttataa agtcgtcaat ggcatgacgc acaatcggtccttcaaccat tgtaaaacca acgtcgaatt tgcataacgtgtcgtcctca acatgattag tgacacgata agcgacaaaaatttcgggaa aatttagttg ttgatcctca acatcgtccacttcgggtac gtttcatata cgtttatttt ggacaaccta a

DESCRIPTION OF THE FIGURES

FIG. 1: Shown are the average symptom levels (of three replicates) at 35days post infection (dpi) with ToLCNDV by whitefly transmission of adonor plant line being ToLCNDV resistant (Wild Donor), a recurrent plantline (Recurrent) and of plant line NCIMB 42625. The average symptomlevels were determined as described herein under “General Methods”.

GENERAL METHODS

1. Determination of Symptom Level on ToLCNDV Infected Plants

1.1 Plants and Pathogens (Virus)

A melon plant (Cucumis melo) infecting strain of ToLCNDV was used forinfection of melon plants. In the present invention a ToLCNDV strainisolated in Murcia, Spain was used as inoculum.

1.2 ToLCNDV Inoculum

The ToLCNDV inoculum source was maintained on living infected melonplants. It must be ensured, that pure virus isolates are used and thatneither the virus source, nor the whiteflies are contaminated with otherdiseases, in particular with other viruses (e.g. CGMMV, CYSDV, CYVY,SqMV). For pre-multiplication of the ToLCNDV inoculum whiteflies(Bemisia tabaci) were fed on ToLCNDV sensitive (susceptible), infectedmelon plants in an insect proof cage. Before infection of test plants,the ToLCNDV infected plants were placed into an insect proof cage,whiteflies were released into the same cage and allowed to feed forapproximately 3 days on the ToLCNDV infected plants.

1.3 Inoculation of Plants to be Tested

For each genotype of melon plants to be analysed 14 plants were grownuntil the first true leaf is expanded (normally 12-15 days aftersowing), 12 of which were infected and 2 were mock infected. Also 12plants of susceptible varieties were included, in this experimentvariety Gandalf F1 (Hild Samen) and variety Vedantrais. The 12 plantsper genotype to be tested for ToLCNDV resistance were placed into aninsect proof cage, infected whiteflies (obtained as described under 1.2above) were released into the cage to infect the plants. It has to beensured that at least 5-10 whiteflies are available for each test plantin the cage. Whiteflies and test plants are kept in the cage forapproximately 48 hours, then the whiteflies are eliminated with anappropriate insecticide. Also two plants per genotype were mockinfected, i.e. they were treated in the same manner as the test plantsapart from the fact that the whiteflies used for infection were free ofToLCNDV.

1.4 Growing Infected Test Plants

Infected test plants obtained as described under 1.3 were transplantedto bigger pots, transferred into a greenhouse with cooling equipment.The plants were grown at approximately 18° C. night temperature andapproximately 25° C. day temperature in a timeframe of 14 to 16 hoursdaylight. The infected plants for each infected genotype were grown intwo replicates in two different plots, each of which comprised 6 ToLCNDVinfected plants and 1 mock infected plant. The plots are randomized inrespect to the growing area.

1.5. Scoring the Symptom Level of ToLCNDV Infection

The scoring of the symptom level may already be done approximately 15days post infection (dpi) with ToLCNDV but is preferably doneapproximately 30 days post infection (dpi) with ToLCNDV, or later, e.g.35 dpi. In case plants are present which show recovery from the virusinfection, a further scoring of the symptom can optionally be doneapproximately 45 days post infection (dpi) with ToLCNDV.

The following symptom levels are to be used according to the phenotypesindicated in the following:

Symptom level Observed phenotype 1 Dead plant 2 Severe mosaic andcurling, chlorosis and growth reduction. No recovery 3 Strong mosaic andcurling, chlorosis and growth reduction. No recovery 4 Curling andmosaic, chlorosis, no or mild growth reduction. No recovery 5 Curlingand mosaic, chlorosis, no growth reduction. Slight recovery of the upperplant zone 6 Mild curling, mosaic and chlorosis, no growth reduction.Recovery of the upper middle plant 7 Mild curling, mosaic and chlorosis,no growth reduction. Symptoms appear only in the lower plant zone 8Faint mosaic 9 No symptoms

1.6 Optional Additional Tests

It is recommended to use at least one genotype highly resistant toToLCNDV (e.g. average symptom level 8-9) and/or one genotype highlysensitive to ToLCNDV (e.g. average symptom level 1 or 2) in eachexperimental setup as controls. It is further recommended to alsoinclude a genotype having intermediate resistance to ToLCNDV infectionin each test setup. When these control genotypes show the expectedsymptom level, this gives a clear indication that the experimentalconditions are right to evaluate the test plants. It is especiallyimportant to include a susceptible control line or variety, which showsthe expected symptoms at the time of scoring, e.g. 30 dpi or later (e.g.35 dpi).

Furthermore, it is advisable to check infection and spreading of ToLCNDVin infected plants and control plants. This can be done by checking forthe presence and/or amount of virus DNA in upper parts of the plants. Asuitable way to check for the presence and/or amount of ToLCNDV DNA inupper plant parts is hybridization of plant material with a probehybridizing with the DNA of the ToLCNDV strain used. Varioushybridization techniques are well known in the art. A simple so calledDot Blot analysis is sufficient for obtaining valuable results.Additionally or alternatively qPCR can be used.

1. Selection of ToLCNDV Resistant Donor Plants

The symptom level of wild accessions of melon plants were tested forToLCNDV resistance according to the test described under “GeneralMethods”. A wild donor plant was identified which has a high resistanceto ToLCNDV infection, having an average disease score of 9 (no symptoms)at 15 dpi, 25 dpi and 35 dpi, while the susceptible control line had anaverage disease score of 2.8 at 15 dpi, 2.0 at 25 dpi and 2.0 at 35 dpi.

Also-blot hybridization and qPCR (quantitative PCR) were carried out oninfected donor plants and a susceptible control line after 30 dpi. NoToLCNDV virus was detected in the donor tissue at 30 dpi, while ToLCNDVvirus was detected in the susceptible control. (Note that a low Ct-valueindicates a high virus titer, while a high Ct-value indicates a lowamount of virus)

qPCR- Dot-blot average Ct signal value ng/μl Donor line − 33.8 3.76E−07Susceptible line + 13.1 0.19 qPCR negative control Ct = 34

Another real time qPCR (quantitative PCR) experiment was carried out,using a protocol described in Simon et al. 2018 (supra). In short, theprimers and probes ToLA-up (forward primer), ToLA-Low (reverse primer)and ToLA-Probe (TaqMan probe) of Table 1 of Simon et al 2018 (supra)were used in a qPCR analysis as described therein, with thermos cyclingconditions being incubation at 95° C. for 2 minutes followed by 40cycles of 95° C. for 15 seconds and 60° C. for 1 minute.

Plants of the donor and of the susceptible variety Vedantrais were sownand inoculated with ToLCNDV as described herein above. Three replicatesof six plants per genotype were used and three mock inoculatedVedantrais plants were included per replicate. Phenotypic scoring ofeach plant was done at 15 dpi, 30 dpi and 45 dpi. Samples for qPCR weretaken at 60 dpi from each plant.

Average Average ToLCNDV Ct-value Genotype disease score STDV (60 dpi)STDV Donor line 9.0 0.0 30.59 3.12 Susceptible line  2.75  0.49 10.071.32 (Vedantrais) Mock inoculated 9.0 0.0 39.97 0.42 susceptible line(Vedantrais) STDV = standard deviation

Results confirmed that the symptom free donor line had extremely lowToLCNDV virus levels in the leaves at 60 dpi.

2. Identification of Genomic Location of ToLCNDV Resistance

Three mapping populations were developed using the donor plant obtainedin Example 1 to map the position of the ToLCNDV resistance conferringloci (QTL). Phenotyping of the mapping populations was carried out asdescribed above, using a TolCNDV scale of 1 to 9. Genotyping wasinitially carried out using 192 SNP markers, and later additional SNPmarkers were run to saturate the QTL regions.

Analysis in these mapping populations revealed two major QTLs associatedwith resistance, located on chromosome 11 and 12.

From resistant material the inventors developed backcross lines to finemap and further investigate resistance from the donor.

The markers identified during fine mapping and their respectivepositions according to publicly known data from Diaz et al. (2015, MolBreeding 35, 188) is shown in Table 2 (in the description).

-   3. Development of KASP-Assays

A KASP-assay was developed for identifying the SNPs linked to QTL11(SNP_01, SNP_02, SNP_03 and SNP_04) and QTL12 (SNP_05, SNP_06 andSNP_07). The SNPs associated with QTL11 and QTL12 can be determined byuse of the following primers in a KASP-assay:

SNP FAM allele VIC allele Common Primer SNP_01 SEQ ID NO 8 SEQ ID NO 9SEQ ID NO 10 SNP_02 SEQ ID NO 11 SEQ ID NO 12 SEQ ID NO 13 SNP_03 SEQ IDNO 14 SEQ ID NO 15 SEQ ID NO 16 SNP_04 SEQ ID NO 17 SEQ ID NO 18 SEQ IDNO 19 SNP_05 SEQ ID NO 20 SEQ ID NO 21 SEQ ID NO 21 SNP_06 SEQ ID NO 23SEQ ID NO 24 SEQ ID NO 25 SNP_07 SEQ ID NO 26 SEQ ID NO 27 SEQ ID NO 28

4. Introgression of ToLCNDV into a Cultivated Melon Plant

Backcrossing was performed with a recurrent parent elite breeding lineof a Piel de Sapo type to develop a line comprising both QTL11 and QTL12in homozygous form.

Seeds of an introgression line comprising both QTL11 and QTL12 inhomozygous form were deposited under NCIMB 42625. The donor genotype ispresent for SNP_01 to SNP_04 and for SNP_05 to SNP_07, as verified usingthe KASP assay.

FIG. 1 and the Table below shows that the donor and the introgressionline deposited have an average disease score of 9.0 when scored 35 dpi,while the susceptible recurrent parent line has an average disease scoreof 2.4 at 35 dpi.

AVG STDV Wild Donor 9.0 0.0 Recurrent (susceptible) parent 2.4  0.43NCIMB 42625 (melon plant comprising 9.0 0.0 introgression fragment onchr. 11 and chr. 12) AVG: average value of tested genotype, STDV:standard deviation

Additional backcrossing was done with the donor to introgress one orboth QTLs into elite Galia, Cantaloup and Charentais backgrounds.

1. A cultivated melon plant or plant cell comprising an introgressionfragment from chromosome 11 of a ToLCNDV resistant donor plant, whereinthe introgression fragment on chromosome 11 comprises a sequence of theToLCNDV resistant donor melon plant in-between SNP_01 at nucleotide 101of SEQ ID NO: 1 and SNP_04 at nucleotide 101 of SEQ ID NO: 4, andwherein the introgression fragment comprises a QTL which confers ToLCNDVresistance in a recessive manner and wherein the introgression fragmentcomprises a Thymine for SNP_03 at nucleotide 101 of SEQ ID NO:
 3. 2. Thecultivated melon plant or plant cell according to claim 1, wherein theintrogression fragment on chromosome 11 further comprises a Thymine atnucleotide 101 of SEQ ID NO: 4 and/or a Guanine at nucleotide 101 of SEQID NO:
 2. 3. A cultivated melon plant or plant cell according to claim1, wherein the introgression fragment on chromosome 11 comprises aAdenine at nucleotide 101 of SEQ ID NO: 1 or of a sequence comprising atleast 90% sequence identity to SEQ ID NO: 1, and a Guanine at nucleotide101 of SEQ ID NO: 2 or of a sequence comprising at least 90% sequenceidentity to SEQ ID NO:2 and a Thymine at nucleotide 101 of SEQ ID NO: 3or of a sequence comprising at least 90% sequence identity to SEQ IDNO:3 and a Thymine at nucleotide 101 of SEQ ID NO: 4 or of a sequencecomprising at least 90% sequence identity to SEQ ID NO:4.
 4. Thecultivated melon plant or plant cell according to claim 1, wherein theintrogression fragment on chromosome 11 is in homozygous form.
 5. Thecultivated melon plant or plant cell according to claim 1, wherein theintrogression fragment on chromosome 11 is from a wild melon or wildrelative of melon.
 6. The cultivated melon plant or plant cell accordingto claim 1, wherein the introgression fragment on chromosome 11comprises a recessive QTL which confers an average TolCNDV resistancescore of at least 7 on a scale of 1 (dead plant) to 9 (no symptoms) whenthe QTL is in homozygous form.
 7. The cultivated melon plant or plantcell according to claim 1, wherein the plant or plant cell is an inbredplant or plant cell or an F1 hybrid plant or plant cell.
 8. A seed whichgrows into a plant according to claim
 1. 9. A cultivated melon fruit orfruit part comprising plant cells according to claim
 1. 10. A cultivatedmelon plant propagation material comprising a cultivated melon plantcell according to claim
 1. 11. A method of crossing an inbred lineaccording to claim 7 to another inbred line and collecting the F1 hybridseed produced from said cross.
 12. A method for producing hybridcultivated melon seeds comprising the following steps a) providing afirst ToLCNDV resistant inbred melon plant comprising two chromosomes 11each having an introgression fragment from chromosome 11 of a ToLCNDVresistant donor plant, the introgression fragment comprising QTL11 and aThymine at nucleotide 101 of SEQ ID NO: 3 for SNP_03; or theintrogression fragment comprising QTL11 and the resistant donor genotypefor one or more or all of SNP_01 at nucleotide 101 of SEQ ID NO: 1,SNP_02 at nucleotide 101 of SEQ ID NO: 2, SNP_03 at nucleotide 101 ofSEQ ID NO: 3 and/or SNP_04 at nucleotide 101 of SEQ ID NO: 4; b)providing a second ToLCNDV resistant inbred melon plant comprising twochromosomes 11 each having an introgression fragment from chromosome 11of a ToLCNDV resistant donor plant, the introgression fragmentcomprising QTL11 and a Thymine at nucleotide 101 of SEQ ID NO: 3 forSNP_03; or the introgression fragment comprising QTL11 and the resistantdonor genotype for one or more or all of SNP_01 at nucleotide 101 of SEQID NO: 1, SNP_02 at nucleotide 101 of SEQ ID NO: 2, SNP_03 at nucleotide101 of SEQ ID NO: 3 and/or SNP_04 at nucleotide 101 of SEQ ID NO: 4; c)crossing the inbred melon plant provided in step a) with the inbredmelon plant provided in step b); and d) collecting seeds obtained fromthe cross of step c). 13-14. (canceled)
 15. A method of screening plantsor plant parts, or DNA derived therefrom, for the presence of a fragmenton chromosome 11 conferring ToLCNDV resistance comprises the steps of:i) screening the genomic DNA for the SNP genotype of one or more or allof SNP_01 01 at nucleotide 101 of SEQ ID NO: 1, SNP_02 at nucleotide 101of SEQ ID NO: 2, SNP_03 at nucleotide 101 of SEQ ID NO: 3 and/or SNP_04at nucleotide 101 of SEQ ID NO: 4; ii) and optionally selecting a plantor plant part comprising an Adenine at nucleotide 101 of SEQ ID NO: 1and/or a Guanine at nucleotide 101 of SEQ ID NO: 2 and/or a Thymine atnucleotide 101 of SEQ ID NO: 3 and/or a Thymine at nucleotide 101 of SEQID NO:
 4. 16. The cultivated melon plant or plant cell according toclaim 1, wherein the donor plant is not Ag-WM7Ind/WM7.